Human sel-10 polypeptides and polynucleotides that encode them

ABSTRACT

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding either of two alternative splice variants of human sel-10, one of which is expressed in hippocampal cells, and one of which is expressed in mammary cells. The invention also provides isolated sel-10 polypeptides and cell lines which express them in which Aβ processing is altered.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following provisional application: U.S. Ser. No. 60/068,243, now abandoned, filed Dec. 19, 1997, under 35 USC 119(e)(1). This application is a continuation-in-part of the following application: U.S. Ser. No. 09/213,888, filed Dec. 17, 1998, under 35 USC 120.

FIELD OF THE INVENTION

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding either of two alternative splice variants of human sel-10, one of which is expressed in hippocampal cells, and one of which is expressed in mammary cells. The invention also provides isolated sel-10 polypeptides.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a degenerative disorder of the central nervous system which causes progressive memory and cognitive decline during mid to late adult life. The disease is accompanied by a wide range of neuropathologic features including extracellular amyloid plaques and intra-neuronal neurofibrillary tangles. (Sherrington, R., et al.; Nature 375: 754-60 (1995)). Although the pathogenic pathway leading to AD is not well understood, several genetic loci are known to be involved in the development of the disease.

Genes associated with early onset Alzheimer's disease (AD) have been identified by the use of mapping studies in families with early-onset AD. These studies have shown that genetic loci on chromosomes 1 and 14 were likely to be involved in AD. Positional cloning of the chromosome 14 locus identified a novel mutant gene encoding an eight-transmembrane domain protein which subsequently was named presenilin-1 (PS-1). (Sherrington, R., et al.; Nature 375: 754-60 (1995)). Blast search of the human EST database revealed a single EST exhibiting homology to PS-1, designated presenilin-2 (PS-2) which was shown to be the gene associated with AD on chromosome 1. (Levy-Lahad, E. et al., Science 269:973-977 (1995); Rogaev, E. I., et al., Nature 376: 775-8 (1995); Li, J. et al., Proc. Natl. Acad. Sci. U.S.A. 92: 12180-12184 (1995)).

Mutations in PS-1 and PS-2 that are associated with Alzheimer's disease are primarily missense mutations. Both PS-1 and PS-2 undergo proteolytic processing, which can be altered by the point mutations found in familial Alzheimer's disease [Perez-Tur, J. et al., Neuroreport 7: 297-301 (1995); Mercken, M. et al., FEBS Lett. 389: 297-303 (1996)]. PS-1 gene expression is widely distributed across tissues, while the highest levels of PS-2 mRNA are found in pancreas and skeletal muscle. (Li, J. et al., Proc. Natl. Acad. Sci. U.S.A. 92: 12180-12184 (1995); Jinhe Li, personal communication). The highest levels of PS-2 protein, however, are found in brain (Jinhe Li, personal communication). Both PS-1 and PS-2 proteins have been localized to the endoplasmic reticulum, the Golgi apparatus, and the nuclear envelope. (Jinhe Li, personal communication; Kovacs, D. M. et al., Nat. Med. 2:224-229 (1996); Doan, A. et al., Neuron 17: 1023-1030 (1996)). Mutations in either the PS-1 gene or the PS-2 gene alter the processing of the amyloid protein precursor (APP) such that the ratio of A-beta₁₋₄₂ is increased relative to A-beta₁₋₄₀ (Scheuner, D. et al., Nat. Med. 2: 864-870 (1996)). When coexpressed in transgenic mice with human APP, a similar increase in the ratio of A-beta₁₋₄₂ as compared to A-beta₁₋₄₀ is observed (Borchelt, D. R. et al., Neuron 17: 1005-1013 (1996); Citron, M. et al., Nat. Med. 3: 67-72 (1997); Duff, K. et al., Nature 383: 710-713 (1996)), together with an acceleration of the deposition of A-beta in amyloid plaques (Borchelt et al., Neuron 19: 939 (1997).

Despite the above-described observations made with respect to the role of PS-1 and PS-2 in AD, their biological function remains unknown, placing them alongside a large number of human disease genes having an unknown biological function. Where the function of a gene or its product is unknown, genetic analysis in model organisms can be useful in placing such genes in known biochemical or genetic pathways. This is done by screening for extragenic mutations that either suppress or enhance the effect of mutations in the gene under analysis. For example, extragenic suppressors of loss-of-function mutations in a disease gene may turn on the affected genetic or biochemical pathway downstream of the mutant gene, while suppressers of gain-of-function mutations will probably turn the pathway off.

One model organism that can be used in the elucidation of the function of the presenilin genes is C. elegans, which contains three genes having homology to PS-1 and PS-2, with sel-12 having the highest degree of homology to the genes encoding the human presenilins. Sel-12 was discovered in a screen for genetic suppressers of an activated notch receptor, lin-12(d) (Levitan, D. et al., Nature 377: 351-354 (1995)). Lin-12 functions in development to pattern cell lineages. Hypermorphic mutations such as lin-12(d), which increase lin-12 activity, cause a “multi-vulval” phenotype, while hypomorphic mutations which decrease activity cause eversion of the vulva, as well as homeotic changes in several other cell lineages (Greenwald, I., et al., Nature 346: 197-199 (1990); Sundaram, M. et al., Genetics 135: 755-763 (1993)). Sel-12 mutations suppress hypermorphic lin-12(d) mutations, but only if the lin-12(d) mutations activate signaling by the intact lin-12(d) receptor (Levitan, D. et al., Nature 377: 351-354 (1995)). Lin-12 mutations that truncate the cytoplasmic domain of the receptor also activate signaling (Greenwald, I., et al., Nature 346: 197-199 (1990)), but are not suppressed by mutations of sel-12 (Levitan, D. et al., Nature 377: 351-354 (1995)). This implies that sel-12 mutations act upstream of the lin-12 signaling pathway, perhaps by decreasing the amount of functional lin-12 receptor present in the plasma membrane. In addition to suppressing certain lin-12 hypermorphic mutations, mutations to sel-12 cause a loss-of-function for egg laying, and thus internal accumulation of eggs, although the mutants otherwise appear anatomically normal (Levitan, D. et al., Nature 377: 351-354 (1995)). Sel-12 mutants can be rescued by either human PS-1 or PS-2, indicating that sel-12, PS-1 and PS-2 are functional homologues (Levitan, D., et al., Proc. Natl. Acad. Sci. U.S.A. 93: 14940-14944 (1996)).

A second gene, sel-10, has been identified in a separate genetic screen for suppressors of lin-12 hypomorphic mutations. Loss-of-function mutations in sel-10 restore signaling by lin-12 hypomorphic mutants. As the lowering of sel-10 activity elevates lin-12 activity, it can be concluded that sel-10 acts as a negative regulator of lin-12 signaling. Sel-10 also acts as a negative regulator of sel-12, the C. elegans presenilin homologue (Levy-Lahad, E. et al., Science 269:973-977 (1995)). Loss of sel-10 activity suppresses the egg laying defect associated with hypomorphic mutations in sel-12 (Iva Greenwald, personal communication). The effect of loss-of-function mutations to sel-10 on lin-12 and sel-12 mutations indicates that sel-10 acts as a negative regulator of both lin-12/notch and presenilin activity. Thus, a human homologue of C. elegans sel-10 would be expected to interact genetically and/or physiologically with human presenilin genes in ways relevant to the pathogenesis of Alzheimer's Disease.

In view of the foregoing, it will be clear that there is a continuing need for the identification of genes related to AD, and for the development of assays for the identification of agents capable of interfering with the biological pathways that lead to AD.

INFORMATION DISCLOSURE

Hubbard E J A, Wu G, Kitajewski J, and Greenwald I (1997) Sel-10, a negative regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins. Genes & Dev 11:3182-3193.

Greenwald-I; Seydoux-G (1990) Analysis of gain-of-function mutations of the lin-12 gene of Caenorhabditis elegans. Nature. 346: 197-9

Kim T-W, Pettingell W H, Hallmark O G, Moir R D, Wasco W, Tanzi R (1997) Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. J Biol Chem 272:11006-11010.

Levitan-D; Greenwald-I (1995) Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene. Nature. 377: 351-4.

Levitan-D; Doyle-T G; Brousseau-D; Lee-M K; Thinakaran-G; Slunt-H H; Sisodia-S S; Greenwald-I (1996) Assessment of normal and mutant human presenilin function in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 93: 14940-4.

Sundaram-M; Greenwald-I (1993) Suppressors of a lin-12 hypomorph define genes that interact with both lin-12 and glp-1 in Caenorhabditis elegans. Genetics. 135: 765-83.

Sundaram-M; Greenwald-I (1993) Genetic and phenotypic studies of hypomorphic lin-12 mutants in Caenorhabditis elegans. Genetics. 135: 755-63.

F55B12.3 GenPep Report (WMBL locus CEF55B12, accession z79757).

WO 97/11956

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding human sel-10, which is expressed in hippocampal cells and in mammary cells. Unless otherwise noted, any reference herein to sel-10 will be understood to refer to human sel-10, and to encompass both hippocampal and mammary sel-10. Fragments of hippocampal sel-10 and mammary sel-10 are also provided.

In a preferred embodiment, the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a human sel-10 polypeptide having the complete amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or as encoded by the cDNA clone contained in ATCC Deposit No.98978;

(b) a nucleotide sequence encoding a human sel-10 polypeptide having the complete amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or as encoded by the cDNA clone contained in ATCC Deposit No. 98979; and

(c) a nucleotide sequence complementary to the nucleotide sequence of (a) or (b).

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide encoding sel-10, or fragments thereof.

The present invention also provides vectors comprising the isolated nucleic acid molecules of the invention, host cells into which such vectors have been introduced, and recombinant methods of obtaining a sel-10 polypeptide comprising culturing the above-described host cell and isolating the sel-10 polypeptide.

In another aspect, the invention provides isolated sel-10 polypeptides, as well as fragments thereof. In a preferred embodiment, the sel-10 polypeptides have an amino acid sequence selected from the group consisting of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, and 10. Isolated antibodies, both polyclonal and monoclonal, that bind specifically to sel-10 polypeptides are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: FIGS. 1A and 1B are western blots showing protein expression in HEK293 cells transfected with PS1-C-FLAG, 6-myc-N-sel-10, and APP695NL-KK cDNAs.

FIGS. 2A and 2B: FIGS. 2A and 2B are northern blots. FIG. 2A is a multiple tissue northern blot probed with the common, mammary sel-10 mRNA showing ubiquitous expression of the 6.5- and 4.5-Kb transcripts. FIG. 2B is a multiple tissue northern blot showing limited expression of the hippocampal sel-10 mRNA only in brain.

FIGS. 3A, 3B and 3C. FIGS. 3A, 3B and 3C are western blots. FIG. 3A demonstrates that SEL-10-myc forms a complex with PS1 as shown by immunoprecipitation with anti-PS1 loop antibody. HEK293 cells were transfected with constructs containing SEL-10-myc or PS1, cotransfected with both constructs, transfected with the corresponding vector only control (pcDNA3 or pCS, respectively), or mock transfected without DNA. Cultures were treated with lactacystin (12 μM) to inhibit proteasome function as indicated. The immunoprecipitates were loaded on the left side of the gel and cell lysates on the right. Controls for immunoprecipitation were a nonspecific IgG and just anti-PS1 loop antibody with protein G beads with no cell lysate (“no protein”). The immunoblot was probed with anti-myc antibody to detect SEL-10-myc in the immunoprecipitates and cell lysates. SEL-10-myc is expressed in lysates from cells transfected with SEL-10-myc or cotransfected with SEL-10-myc and PS1, but was detected only as a complex with PS1 in cells cotransfected with the SEL-10-myc and PS1 constructs. FIG. 3B demonstrates that the SEL-10-myc/PS1 complex could be detected only when proteasome degradation was inhibited with lactacystin. FIG. 3C demonstrates that immunoprecipitation of the SEL-10-myc/PS1 complex with anti-myc antibody indicates that SEL-10-myc associates primarily with full length and high molecular weight forms of PS1. Immunoprecipitates were probed with either anti-PS1 loop or anti-myc antibodies. Longer exposures of the ECL developed Western blot reveal very low levels of PS1 -NTF in the immunoprecipitate with SEL-10-myc, but fail to detect PS1-CTF.

FIGS. 4A and 4B. FIGS. 4A and 4B are western blots. FIG. 4A demonstrates that SEL-10-myc stimulates ubiquitination of PS1. An HEK293 cell line with stable expression of PS1 was transfected with SEL-10-myc. Immunoprecipitation with an anti-PS1 loop antibody was followed by detection with an anti-ubiquitin antibody. FIG. 4B demonstrates that increased expression of SEL-10-myc leads to a decrease in PS1-NTF and PS1-CTF with a corresponding increase in the amount of full length PS1. Cell lysates were immunoprecipitated with anti-PS1 loop antibody and then probed on the Western blot with the same antibody to detect PS1 and its processing products.

FIGS. 5A, 5B and 5C. FIGS. 5A, 5B and 5C are graphs. FIG. 5A demonstrates that transient co-expression of SEL-10-myc with APP increases production of Aβ1-40 and Aβ1-42. The effect is additive with co-expressed PS1. FIG. 5B demonstrates that stable expression of SEL-10-myc or PS1 increases endogenous Aβ production. FIG. 5C demonstrates that transient expression of APP in the stable cell lines increases exogenous Aβ production over the level seen in a cell line transformed with the pcDNA3.1 vector control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding human sel-10. The nucleotide sequence of human hippocampal sel-10 (hhsel-10), which sequence is given in SEQ ID NO:1, encodes five hhsel-10 polypeptides (hhsel-10-(1), hhsel-10-(2), hhsel-10-(3), hhsel-10-(4), and hhsel-10-(5), referred to collectively herein as hhsel-10). The nucleotide sequence of human mammary sel-10 (hmsel-10), which sequence is given in SEQ ID NO:2, encodes three hmsel-10 polypeptides (hmSel-10-(1), hmSel-10-(2), and hmsel-10-(3), referred to collectively herein as hmsel-10). The nucleotide sequences of the hhsel-10 polynucleotides are given in SEQ ID NO. 1, where nucleotide residues 45-1928 of SEQ ID NO. 1 correspond to hhsel-10-(1), nucleotide residues 150-1928 of SEQ ID NO. 1 correspond to hhSel-10-(2), nucleotide residues 267-1928 of SEQ ID NO. 1 correspond to hhSel-10-(3), nucleotide residues 291-1928 of SEQ ID NO. 1 correspond to hhSel-10-(4), and nucleotide residues 306-1928 of SEQ ID NO. 1 correspond to hhSel-10-(5). The nucleotide sequences of the hmSel-10 polynucleotides are given in SEQ ID NO. 2, where nucleotide residues 180-1949 of SEQ ID NO. 2 correspond to hmSel-10-(1), nucleotide residues 270-1949 of SEQ ID NO. 2 correspond to hmSel-10-(2), and nucleotide residues 327-1949 of SEQ ID NO. 2 correspond to hmSel-10-(3). The amino acid sequences of the polypeptides encoded by the hhSel-10 and hm-Sel-10 nucleic acid molecules are given as follows: SEQ ID NOS: 3, 4, 5, 6, and 7 correspond to the hhSel-10-(1), hhSel-10-(2), hhSel-10-(3). hhSel-10-(4), and hhSel-10-(5) polypeptides, respectively, and SEQ ID NOS: 8, 9, and 10 correspond to the hmSel-10-(1), hmSel-10-(2), and hmSel-10-(3) polypeptides, respectively. Unless otherwise noted, any reference herein to sel-10 will be understood to refer to human sel-10, and to encompass all of the hippocampal and mammary sel-10 nucleic acid molecules (in the case of reference to sel-10 nucleic acid, polynucleotide, DNA, RNA, or gene) or polypeptides (in the case of reference to sel-10 protein, polypeptide, amino acid sequence). Fragments of hippocampal sel-10 and mammary sel-10 nucleic acid molecules and polypeptides are also provided.

The nucleotide sequence of SEQ ID NO:1 was obtained as described in Example 1, and is contained in cDNA clone PNV 102-1, which was deposited on Nov. 9, 1998, at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110, and given accession number 98978. The nucleotide sequence of SEQ ID NO:2 was obtained as described in Example 1, and is contained in cDNA clone PNV 108-2, which was deposited on Nov. 9, 1998, at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110, and given accession number 98979.

The human sel-10 polypeptides of the invention share homology with C. elegans sel-10, as well as with members of the β-transducin protein family, including yeast CDC4, and human LIS-1. This family is characterized by the presence of an F-box and multiple WD-40 repeats (Li, J., et al., Proc. Natl. Acad. Sci. U.S.A. 92:12180-12184 (1995)). The repeats are 20-40 amino acids long and are bounded by gly-his (GH) and trp-asp (WD) residues. The three dimensional structure of β-transducin indicates that the WD40 repeats form the arms of a seven-bladed propeller like structure (Sondek, J., et al., Nature 379:369-374 (1996)). Each blade is formed by four alternating pleats of beta-sheet with a pair of the conserved aspartic acid residues in the protein motif forming the limits of one internal beta strand. WD40 repeats are found in over 27 different proteins which represent diverse functional classes (Neer, E. J., et al., Nature 371:297-300 (1994)). These regulate cellular functions including cell division, cell fate determination, gene transcription, signal transduction, protein degradation, mRNA modification and vesicle fusion. This diversity in function has led to the hypothesis that β-transducin family members provide a common scaffolding upon which multiprotein complexes can be assembled.

The nucleotide sequence given in SEQ ID NO:1 corresponds to the nucleotide sequence encoding hhsel-10, while the nucleotide sequence given in SEQ ID NO:2 corresponds to the nucleotide sequence encoding hmsel-10. The isolation and sequencing of DNA encoding sel-10 is described below in Examples 1 and 2.

As is described in Examples 1 and 2, automated sequencing methods were used to obtain the nucleotide sequence of sel-10. The sel-10 nucleotide sequences of the present invention were obtained for both DNA strands, and are believed to be 100% accurate. However, as is known in the art, nucleotide sequence obtained by such automated methods may contain some errors. Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art. An error in sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation. The sel-10 DNA of the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic sel-10 DNA may be obtained by screening a genomic library with the sel-10 cDNA described herein, using methods that are well known in the art. RNA transcribed from sel-10 DNA is also encompassed by the present invention.

Due to the degeneracy of the genetic code, two DNA sequences may differ and yet encode identical amino acid sequences. The present invention thus provides isolated nucleic acid molecules having a polynucleotide sequence encoding any of the sel-10 polypeptides of the invention, wherein said polynucleotide sequence encodes a sel-10 polypeptide having the complete amino acid sequence of SEQ ID NOs:3-10, or fragments thereof.

Also provided herein are purified sel-10 polypeptides, both recombinant and non-recombinant. Variants and derivatives of native sel-10 proteins that retain any of the biological activities of sel-10 are also within the scope of the present invention. As is described above, the sel-10 polypeptides of the present invention share homology with yeast CDC4. As CDC4 is known to catalyze ubiquitination of specific cellular proteins (Feldman et al., Cell 91:221 (1997)), it may be inferred that sel-10 will also have this activity. Assay procedures for demonstrating such activity are well known, and involve reconstitution of the ubiquitinating system using purified human sel-10 protein together with the yeast proteins Cdc4p, Cdc53p and Skp1p, or their human orthologs, and an E1 enzyme, the E2 enzyme Cdc34p or its human ortholog, ubiquitin, a target protein and an ATP regenerating system (Feldman et al., 1997). Skp1p associates with Cdc4p through a protein domain called an F-box (Bai et al., Cell 86:263 (1996)). The F-box protein motif is found in yeast CDC4, C. elegans sel-10, mouse sel-10 and human sel-10. The sel-10 ubiquitination system may be reconstituted with the C. elegans counterparts of the yeast components, e.g., cul-1 (also known as lin-19) protein substituting for Cdc53p (Kipreos et al., Cell 85:829 (1996)) and the protein F46A9 substituting for Skp1p, or with their mammalian counterparts, e.g., Cul-2 protein substituting for Cdc53p (Kipreos et al., 1996) and mammalian Skp1p substituting for yeast Skp1p. A phosphorylation system provided by a protein kinase is also included in the assay system as per Feldman et al., 1997.

Sel-10 variants may be obtained by mutation of native sel-10-encoding nucleotide sequences, for example. A sel-10 variant, as referred to herein, is a polypeptide substantially homologous to a native sel-10 but which has an amino acid sequence different from that of native sel-10 because of one or more deletions, insertions, or substitutions in the amino acid sequence. The variant amino acid or nucleotide sequence is preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical, to a native sel-10 sequence. Thus, a variant nucleotide sequence which contains, for example, 5 point mutations for every one hundred nucleotides, as compared to a native sel-10 gene, will be 95% identical to the native protein. The percentage of sequence identity, also termed homology, between a native and a variant sel-10 sequence may also be determined, for example, by comparing the two sequences using any of the computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), which uses the algorithm of Smith and Waterman (Adv. Appl. Math. 2: 482-489 (1981)).

Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques. For example, mutations may be introduced at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis, which is described by Walder et al. (Gene 42:133 (1986)); Bauer et al. (Gene 37:73 (1985)); Craik (BioTechniques, January 1985, pp. 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press (1981)); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Sel-10 variants within the scope of the invention may comprise conservatively substituted sequences, meaning that one or more amino acid residues of a sel-10 polypeptide are replaced by different residues that do not alter the secondary and/or tertiary structure of the sel-10 polypeptide. Such substitutions may include the replacement of an amino acid by a residue having similar physicochemical properties, such as substituting one aliphatic residue (Ile, Val, Leu or Ala) for another, or substitution between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Further information regarding making phenotypically silent amino acid exchanges may be found in Bowie et al., Science 247:1306-1310 (1990). Other sel-10 variants which might retain substantially the biological activities of sel-10 are those where amino acid substitutions have been made in areas outside functional regions of the protein.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a portion of the nucleic acid molecules described above, e.g., to at least about 15 nucleotides, preferably to at least about 20 nucleotides, more preferably to at least about 30 nucleotides, and still more preferably to at least about from 30 to at least about 100 nucleotides, of one of the previously described nucleic acid molecules. Such portions of nucleic acid molecules having the described lengths refer to, e.g., at least about 15 contiguous nucleotides of the reference nucleic acid molecule. By stringent hybridization conditions is intended overnight incubation at about 42/C for about 2.5 hours in 6×SSC/0.1% SDS, followed by washing of the filters in 1.0×SSC at 65/C, 0.1% SDS.

Fragments of the sel-10-encoding nucleic acid molecules described herein, as well as polynucleotides capable of hybridizing to such nucleic acid molecules may be used as a probe or as primers in a polymerase chain reaction (PCR). Such probes may be used, e.g., to detect the presence of sel-10 nucleic acids in in vitro assays, as well as in Southern and northern blots. Cell types expressing sel-10 may also be identified by the use of such probes. Such procedures are well known, and the skilled artisan will be able to choose a probe of a length suitable to the particular application. For PCR, 5′ and 3′ primers corresponding to the termini of a desired sel-10 nucleic acid molecule are employed to isolate and amplify that sequence using conventional techniques.

Other useful fragments of the sel-10 nucleic acid molecules are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence capable of binding to a target sel-10 mRNA (using a sense strand), or sel-10 DNA (using an antisense strand) sequence.

In another aspect, the invention includes sel-10 polypeptides with or without associated native pattern glycosylation. Sel-10 expressed in yeast or mammalian expression systems (discussed below) may be similar to or significantly different from a native sel-10 polypeptide in molecular weight and glycosylation pattern. Expression of sel-10 in bacterial expression systems will provide non-glycosylated sel-10.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. Sel-10 polypeptides may be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. In a preferred embodiment, high performance liquid chromatography (HPLC) is employed for purification.

The present invention also relates to vectors comprising the polynucleotide molecules of the invention, as well as host cell transformed with such vectors. Any of the polynucleotide molecules of the invention may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. Because the invention also provides sel-10 polypeptides expressed from the polynucleotide molecules described above, vectors for the expression of sel-10 are preferred. The vectors include DNA encoding any of the sel-10 polypeptides described above or below, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding sel-10. Thus, a promoter nucleotide sequence is operably linked to a sel-10 DNA sequence if the promoter nucleotide sequence directs the transcription of the sel-10 sequence.

Selection of suitable vectors to be used for the cloning of polynucleotide molecules encoding sel-10, or for the expression of sel-10 polypeptides, will of course depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the sel-10 polypeptide is to be expressed. Suitable host cells for expression of sel-10 polypeptides include prokaryotes, yeast, and higher eukaryotic cells, each of which is discussed below.

The sel-10 polypeptides to be expressed in such host cells may also be fusion proteins which include regions from heterologous proteins. Such regions may be included to allow, e.g., secretion, improved stability, or facilitated purification of the polypeptide. For example, a sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in-frame to the sel-10 sequence so that sel-10 is translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cell promotes extracellular secretion of the sel-10 polypeptide. Preferably, the signal sequence will be cleaved from the sel-10 polypeptide upon secretion of sel-10 from the cell. Non-limiting examples of signal sequences that can be used in practicing the invention include the yeast I-factor and the honeybee melatin leader in sf9 insect cells.

In a preferred embodiment, the sel-10 polypeptide will be a fusion protein which includes a heterologous region used to facilitate purification of the polypeptide. Many of the available peptides used for such a function allow selective binding of the fusion protein to a binding partner. For example, the sel-10 polypeptide may be modified to comprise a peptide to form a fusion protein which specifically binds to a binding partner, or peptide tag. Non-limiting examples of such peptide tags include the 6-His tag, thioredoxin tag, FLAG tag, hemaglutinin tag, GST tag, and OmpA signal sequence tag. As will be understood by one of skill in the art, the binding partner which recognizes and binds to the peptide may be any molecule or compound including metal ions (e.g., metal affinity columns), antibodies, or fragments thereof, and any protein or peptide which binds the peptide. These tags may be recognized by fluorescein or rhodamine labeled antibodies that react specifically with each type of tag

Suitable host cells for expression of sel-10 polypeptides include prokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotic hosts to be used for the expression of sel-10 include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. For expression in, e.g., E. coli, a sel-10 polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host. The N-terminal Met may optionally then be cleaved from the expressed sel-10 polypeptide.

Expression vectors for use in prokaryotic hosts generally comprise one or more phenotypic selectable marker genes. Such genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bethesda, Md.), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

Sel-10 may also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.

Vectors replicable in both yeast and E. coli (termed shuttle vectors) may also be used. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coli. Direct secretion of sel-10 polypeptides expressed in yeast hosts may be accomplished by the inclusion of nucleotide sequence encoding the yeast I-factor leader sequence at the 5′ end of the sel-10-encoding nucleotide sequence.

Insect host cell culture systems may also be used for the expression of Sel-10 polypeptides. In a preferred embodiment, the sel-10 polypeptides of the invention are expressed using a baculovirus expression system. Further information regarding the use of baculovirus systems for the expression of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

In another preferred embodiment, the sel-10 polypeptide is expressed in mammalian host cells. Non-limiting examples of suitable mammalian cell lines include the COS-7 line of monkey kidney cells (Gluzman et al., Cell 23:175 (1981)) and Chinese hamster ovary (CHO) cells.

The choice of a suitable expression vector for expression of the sel-10 polypeptides of the invention will of course depend upon the specific mammalian host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and enhancer sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.

The polypeptides of the present invention may also be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting sel-10 polypeptide expression. Such antibodies may be prepared by conventional techniques. See, for example, Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980).

The sel-10 nucleic acid molecules of the present invention are also valuable for chromosome identification, as they can hybridize with a specific location on a human chromosome. There is a current need for identifying particular sites on the chromosome, as few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. The relationship between genes and diseases that have been mapped to the same chromosomal region can then be identified through linkage analysis, wherein the coinheritance of physically adjacent genes is determined. Whether a gene appearing to be related to a particular disease is in fact the cause of the disease can then be determined by comparing the nucleic acid sequence between affected and unaffected individuals.

The sel-10 polypeptides of the invention, and the DNA encoding them, may also be used to further elucidate the biological mechanism of AD, and may ultimately lead to the identification of compounds that can be used to alter such mechanisms. The sel-10 polypeptides of the invention are 47.6% identical and 56.7% similar to C. elegans sel-10. As is described above, mutations to C. elegans sel-10 are known to suppress mutations to sel-12 that result in a loss-of-function for egg laying, and also to suppress certain hypomorphic mutations to lin-12. Mutations to C. elegans sel-12 can also be rescued by either of the human AD-linked genes PS-1 (42.7% identical to sel-12) or PS-2 (43.4% identical to sel-12). However, human PS-1 with a familial AD-linked mutant has a reduced ability to rescue sel-12 mutants (Levitan, D. et al., Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)).

This demonstrated interchangeability of human and C. elegans genes in the notch signaling pathway makes it reasonable to predict that mutations of human sel-10 will suppress mutations to PS-1 or PS-2 that lead to AD, especially in light of the predicted structure of sel-10. As described above, PS-1 and PS-2 mutations that lead to AD are those which interfere with the proteolytic processing of PS-1 or PS-2. The sel-10 polypeptides of the invention are members of the β-transducin protein family, which includes yeast CDC4, a component of an enzyme which functions in the ubiquitin-dependent protein degradation pathway. Thus, human sel-10 may regulate presenilin degradation via the ubiquitin-proteasome pathway. Alternatively, or in addition, human sel-10 may alter presenilin function by targeting for degradation through ubiquitination a modulator of presenilin activity, e.g., a negative regulator. Therefore, mutations to sel-10 may reverse the faulty proteolytic processing of PS-1 or PS-2 which occurs as a result of mutation to PS-1 or PS-2 or otherwise increase presenilin function. For the same reason, inhibition of sel-10 activity may also act to reverse PS-1 or PS-2 mutations. Thus, it may be hypothesized that compounds which inhibit either the expression or the activity of the human sel-10 polypeptides of the invention may reverse the effects of mutations to PS-1 or PS-2, and thus be useful for the prevention or treatment of AD.

Thus, C. elegans may be used as a genetic system for the identification of agents capable of inhibiting the activity or expression of the human sel-10 polypeptides of the invention. A suitable C. elegans strain for use in such assays lacks a gene encoding active C. elegans sel-10, and exhibits a loss-of-function for egg-laying resulting from an inactivated sel-12 gene. Construction of C. elegans strains having a loss-of-function for egg-laying due to mutation of sel-12 may be accomplished using routine methods, as both the sequence of sel-12 (Genebank accession number U35660) and mutations to sel-12 resulting in a loss-of-function for egg laying are known (see Levitan et al., Nature 377: 351-354 (1995), which describes construction of C. elegans sel-12(ar171)). An example of how to make such a strain is also given in Levitan et al. (Nature 377: 351-354 (1995)). Wild-type C. elegans sel-10 in the C. elegans sel-12(ar171)) , is also mutagenized using routine methods, such as the technique used for sel-12 mutagenesis in Levitan et al., supra.

In order to identify compounds inhibiting human sel-10 activity, a DNA vector containing a human sel-10 gene encoding any of the wild-type human sel-10 proteins of the invention is introduced into the above-described C. elegans strain. In a preferred embodiment, the heterologous human sel-10 gene is integrated into the C. elegans genome. The gene is then expressed, using techniques described in Levitan et al. (Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)). Test compounds are then administered to this strain in order to determine whether a given agent is capable of inhibiting sel-10 activity so as to suppress mutations to sel-12 or lin-12 that result in egg-laying defects. Egg-laying in this strain is then determined, e.g. by the assay described in Levitan et al. (Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)). To confirm that the compound's effect on egg-laying is due to inhibition of sel-10 activity, the action of the compound can be tested in a second biochemical or genetic pathway that is known to be affected by loss-of-function mutations in sel-10 (e.g., further elevation of lin-12 activity in lin-12(d) hypomorphic strains). Such assays may be performed as described in Sundarem and Greenwald (Genetics 135: 765-783 (1993)).

Alternatively, compounds are tested for their ability to inhibit the E3 Ubiquitin Ligating Enzyme. Assays procedures for demonstrating such activity are well known, and involve reconstitution of the ubiquitinating system using purified human sel-10 protein together with the yeast proteins Cdc4p, Cdc53p and Skp1p and an E1 enzyme, the E2 enzyme Cdc34p, ubiquitin, a target protein and an ATP regenerating system (Feldman et al., 1997). The sel-10 ubiquitination system may also be reconstituted with the C. elegans counterparts of the yeast components, e.g., cul-1 (also known as lin-19) protein substituting for Cdc53p (Kipreos et al., Cell 85:829 (1996)) and the protein F46A9 substituting for Skp1p, or with their mammalian counterparts, e.g., Cul-2 protein substituting for Cdc53p (Kipreos et al., ibid.) and mammalian Skp1p substituting for yeast Skp1p. A phosphorylation system provided by a protein kinase is also to be included in the assay system as per Feldman et al., 1997.

Alternatively, cell lines which express human sel-10 due to transformation with a human sel-10 cDNA and which as a consequence have elevated APP processing and formation of Aβ₁₋₄₀ or Aβ₁₋₄₂ may also be used for such assays as in Example 3. Compounds may be tested for their ability to reduce the elevated Aβ processing seen in the sel-10 transformed cell line.

Compounds that rescue the egg-laying defect or that inhibit E3 Ubiquitin Ligating Enzyme are then screened for their ability to cause a reduction in the production of A-beta₁₋₄₀ or A-beta₁₋₄₂ in a human cell line. Test compounds are used to expose IMR-32 or other human cell lines known to produce A-beta₁₋₄₀ or A-beta₁₋₄₂ (Asami-Okada et al., Biochemistry 34: 10272-10278 (1995)), or in human cell lines engineered to express human APP at high levels. In these assays, A-beta₁₋₄₀or A-beta₁₋₄₂ is measured in cell extracts or after release into the medium by ELISA or other assays which are known in the art (Borchelt et al., i Neuron 17: 1005-1013 (1996); Citron et al., Nat. Med. 3: 67-72 (1997)).

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLES Example 1

Identification of a Human Homologue to C. elegans sel-10 Results

Identification of sel-10 in ACEDB

Sel-10 maps between the cloned polymorphisms arP3 and TCPARI just to the left of him-5 [ACEDB entry wm95p536]. Three phage lambda clones have been sequenced across the interval, F53C11, F09F3, and F55B12. Sel-10 is reported to have homology to yeast cdc4 [ACEDB entry wm97ab259]. Blast search revealed a single ORF with homology to yeast cdc4 (CC4_YST) within the interval defined by arP3 and TCPARI corresponding to the GenPep entry F55B12.3. F55B12.3, like yeast cdc4, is a member of the β-transducin protein family. This family is characterized by the presence of multiple WD40 repeats [Neer, E. J. et al., Nature 371: 297-300 (1994)].

Identification of a Human sel-10 homologue, Incyte 028971

The GenPep entry F55B12.3 was used to search the LifeSeq, LifeSeq FL and EMBL data bases using tblastn. The search revealed multiple homologies to β-transducin family members including LIS-1 (S36113 and P43035), a gene implicated in Miller-Dieker lissencephaly, a Xenopus laevis gene, TRCPXEN (U63921), and a human contig in LifeSeq FL, 028971. Since there also are multiple β-transducin family members within the C. elegans genome, these were collected using multiple blast searches and then clustered with the sel-10 candidate genes. Multiple alignments were performed with the DNAStar program Megalign using the Clustal method. This revealed that LIS-1 clustered with T03F6.F, a different β-transducin family member and thus excluded it as a candidate sel-10 homologue. TRCPXEN clustered with K10 B2.1, a gene which also clusters with F55B12.3 and CC4YST, while Incyte 028971 clustered with sel-10. Thus, Incyte 028971 appears to encode the human homologue of C. elegans sel-10. Sequence homology between sel-10 and 028971 is strongest in the region of the protein containing 7 repeats of the WD40 motif. The Incyte 028971 contig contains 44 ESTs from multiple libraries including pancreas, lung, T-lymphocytes, fibroblasts, breast, hippocampus, cardiac muscle, colon, and others.

Public EST

Blastx searches with the DNA sequence 028971 against the TREMBLP dataset identified a single homologous mouse EST (W85144) from the IMAGE Library, Soares mouse embryo NbME13.5-14.5. The blastx alignment of 028971 with W85144 and then with F55B12.3 revealed a change in reading frame in 028971 which probably is due to a sequencing error.

Blastn searches of the EMBL EST database with the 028971 DNA sequence revealed in addition to W85144, three human EST that align with the coding sequence of 028971 and six EST that align with the 3′ untranslated region of the 028971 sequence.

Protein Motifs

Two protein motifs were identified in F55B12.3 which are shared with yeast cdc4, mouse w85144 and human 028971. These are an F-box in the N-terminal domain and seven β-transducin repeats in the C-terminal domain.

Discussion

The sel-10 gene encodes a member of the β-transducin protein family. These are characterized by the presence of multiple WD40 repeats [Neer, E. J. et al., Nature 371: 297-300 (1994)]. The repeats are 20-40 amino acids long and are bounded by gly-his (GH) and trp-asp (WD) residues. Solution of the three dimensional structure of β-transducin indicates that the WD40 repeats form the arms of a seven-bladed propeller like structure [Sondek, J. et al., Nature 379: 369-74 (1996)]. Each blade is formed by four alternating pleats of beta-sheet with a pair of the conserved aspartic acid residues in the protein motif forming the limits of one internal beta strand. WD40 repeats are found in over 27 different proteins which represent diverse functional classes [Neer, E. J. et al., Nature 371: 297-300 (1994)]. These regulate cellular functions including cell division, cell fate determination, gene transcription, signal transduction, protein degradation, mRNA modification and vesicle fusion. This diversity in function has led to the hypothesis that β-transducin family members provide a common scaffolding upon which multiprotein complexes can be assembled.

The homology of sel-10, 28971 and W85144 to the yeast cdc4 gene suggests a functional role in the ubiquitin-proteasome pathway for intracellular degradation of protein. Mutations of the yeast cdc4 gene cause cell cycle arrest by blocking degradation of Sic1, an inhibitor of S-phase cyclin/cdk complexes [King, R. W. et al., Science 274: 1652-9 (1996)]. Phosphorylation of Sic1 targets it for destruction through the ubiquitin-proteasome pathway. This pathway consists of three linked enzyme reactions that are catalyzed by multiprotein complexes [Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A. and A. L. Schwartz, FASEB J. 8: 182-91 (1994)]. Initially, the C-terminal glycine of ubiquitin is activated by ATP to form a high energy thiol ester intermediate in a reaction catalyzed by the ubiquitin-activating enzyme, E1. Following activation, an E2 enzyme (ubiquitin conjugating enzyme) transfers ubiquitin from E1 to the protein target. In some cases, E2 acts alone. In others, it acts in concert with an E3 ubiquitin-ligating enzyme which binds the protein substrate and recruits an E2 to catalyze ubiquitination. E2 ubiquitin-conjugating enzymes comprise a fairly conserved gene family, while E3 enzymes are divergent in sequence [Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A. and A. L. Schwartz, FASEB J. 8: 182-91 (1994)].

In yeast, mutation of the E2 ubiquitin-conjugating enzyme, cdc34, causes cell cycle arrest through failure to degrade the Sic1 inhibitor of the S-phase cyclin/cdk complex [King, R. W. et al., Science 274: 1652-9 (1996)]. Sic1 normally is degraded as cells enter the G1-S phase transition, but in the absence of cdc34, Sic1 escapes degradation and its accumulation causes cell cycle arrest. Besides cdc34, cdc4 is one of three other proteins required for the G1-S phase transition. The other two are cdc53 and Skp1. As discussed above, cdc4 contains two structural motifs, seven WD40 repeats (which suggests that the protein forms a beta-propeller) and a structural motif shared with cyclin F which is an interaction domain for Skp1 [Bai, C. et al., Cell 86: 263-74 (1996)]. Insect cell lysates containing cdc53, cdc4 and skp1 (and also ubiquitin, cdc34 and El) can transfer ubiquitin to Sic1 suggesting that one or more of these components functions as an E3 ubiquitin-ligating enzyme [King, R. W. et al., Science 274: 1652-9 (1996)]. Increased expression of either cdc4 or Skp1 partially rescues loss of the other.

In C. elegans, mutation of sel-10 has no visible phenotype indicating that sel-10 does not play a role in regulation of the cell-cycle. A closely related, C. elegans β-transducin family member, K10B2.6 may play that role as it clusters with the gene TRCP_XEN from Xenopus laevis which rescues yeast cell cycle mutants arrested in late anaphase due to a failure to degrade cyclin B [Spevak, W. et al., Mol. Cell. Biol. 13: 4953-66 (1993)]. If sel-10 does encode a component of an E3-ubiquitin ligating enzyme, how might it suppress sel-12 and enhance lin-12 mutations? The simplest hypothesis is that sel-10 regulates degradation of both proteins via the ubiquitin-proteasome pathway. Both sel-12 and lin-12 are transmembrane proteins. Sel-12 crosses the membrane 8 times such that its N- and C-termini face the cytosol [Kim, T. W. et al., J. Biol. Chem. 272: 11006-10 (1997)], while lin-12 is a type 1 transmembrane protein (Greenwald, I. and G. Seydoux, Nature 346: 197-9 (1990)). Both are ubiquitinated, and in the case of human PS2, steady state levels increase in cells treated with an inhibitor of the proteasome, N-acetyl-L-leucinal-L-norleucinal and lactacystin (Li, X. and I. Greenwald, Neuron. 17: 1015-21 (1996)). Alternatively, sel-10 may target for degradation of a negative regulator of presenilin function.

The genetic analysis and protein function suggested by homology to cdc4 implies that drug inhibitors of human sel-10 may increase steady state levels of human presenilins. This could potentiate activity of the presenilin pathway and provide a means for therapeutic intervention in Alzheimer's disease.

Example 2

5′RACE Cloning of a Human cDNA Encoding Sel-10, an Extragenic Suppressor of Presenilin Mutations in C. elegans

Materials and Methods

Oligonucleotide primers for the amplification of the sel-10 coding sequence from C. elegans cDNA were prepared based on the sequence of F55B12.3, identified in Example 1 as the coding sequence for C. elegans sel-10. The primers prepared were: 5′-CGGGATCCACCATGGATGATGGATCGATGACACC-3′ (SEQ ID NO:11) and 5′-GGAATTCCTTAAGGGTATACAGCATCAAAGTCG-3′ (SEQ ID NO:12). C. elegans mRNA was converted to cDNA using a BRL Superscript II Preamplification kit. The PCR product was digested with restriction enzymes BamHI and EcoRI (LTI, Gaithersberg, Md.) and cloned into pcDNA3.1 (Invitrogen). Two isolates were sequenced (ABI, Perkin-Elmer Corp).

The sequence of Incyte clone 028971 (encoding a portion of the human homologue of C. elegans sel-10), was used to design four antisense oligonucleotide primers:5′-TCACTTCATGTCCACATCAAAGTCC-3′ (SEQ ID NO:13), 5′-GGTAA-TTACAAGTTCTTGTTGAACTG (SEQ ID NO:14), 5′-CCCTGCAACGTGTGT-AGACAGG-3′ (SEQ ID NO:15), and 5′-CCAGTCTCTGCATTCCACACTTTG-3′ (SEQ ID NO:16) to amplify the missing 5′ end of human sel-10. The Incyte LifeSeq “Electronic Northern” analysis was used to identify tissues in which sel-10 was expressed. Two of these, hippocampus and mammary gland, were chosen for 5′ RACE cloning using a CloneTech Marathon kit and prepared Marathon-ready cDNA from hippocampus and mammary gland. PCR products were cloned into the TA vector pCR3.1 (Invitrogen), and isolates were sequenced. An alternate 5′ oligonucleotide primer was also designed based on Incyte clones which have 5′ ends that differ from the hippocampal sel-10 sequence: 5′-CTCAGACAGGTCAGGACATTTGG-3′ (SEQ ID NO:17).

Blastn was used to search Incyte databases LifeSeq and LifeSeqFL. Gap alignments and translations were performed with GCG programs (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.).

Results

The Coding Sequence of the C. elegans sel-10

The predicted coding sequence of the C. elegans sel-10, F55B12.3, had originally been determined at the Genome Sequencing Center, Washington University, St. Louis, by using the computer program GeneFinder to predict introns and exons in the genomic cosmid F55B12. The hypothetical cDNA sequence was confirmed by amplifying this region from C. elegans cDNA, cloning, and sequencing it .

The Coding Sequence of the Human sel-10 Gene Homologue

All of the 028971 antisense oligonucleotides amplified a 5′ RACE product from human hippocampal and mammary cDNA. The longest PCR product from the hippocampal reactions was cloned and sequenced. This PCR reaction was designed to generate products which end at the predicted stop codon. Two isolates contained identical sequence which begins 880 bases before the beginning of the 028971 sequence. This sequence was confirmed by comparison with spanning Incyte cDNA clones. The Incyte clones that spanned the 5′ end of the human sel-10 homologue were not annotated as F55B12.3, as the homology in this region between the human and C. elegans genes is low, and as the overlap between these clones and the annotated clones happened to be too small for them to be clustered in the Incyte database or uncovered by our blasting the Incyte database with the 028971 sequence.

The predicted protein sequences of human sel-10 have 47.6% identity and 56.7% similarity to C. elegans sel-10. The N-terminus of the human sel-10 sequence begins with 4 in-frame methionines. In addition to the WD40 repeats described above, the human sequence also contains a region homologous to the CDC4 F-box for binding Skp1, as expected for a sel-10 homologue.

Different Human sel-10 mRNAs Expressed in Mammary and Hippocampal Tissues

Several additional human sel-10 ESTs which differ from the hippocampal sequence were identified. These are an exact match, which indicates that the alternative transcript is probably real. Comparison of these sequences with the human hippocampal sel-10 sequence shows divergence prior to the 4th in-frame methionine and then exact sequence match thereafter. An oligonucleotide primer specific for the 5′ end of this alternative transcript was found to amplify a product from mammary but not hippocampal cDNA. This indicates either that the human sel-10 transcript undergoes differential splicing in a tissue-specific fashion or that the gene contains multiple, tissue specific promoters.

Discussion

5′RACE and PCR amplification were used to clone a full-length cDNA encoding the human homologue of the C. elegans gene, sel-10. Sequence analysis confirms the earlier prediction that sel-10 is a member of the CDC4 family of proteins containing F-Box and WD40 Repeat domains. Two variants of the human sel-10 cDNA were cloned from hippocampus and mammary gland which differed in 5′ sequence preceding the apparent site of translation initiation. This implies that the gene may have two or more start sites for transcription initiation which are tissue-specific or that the pattern of exon splicing is tissue-specific.

Example 3

Expression of Epitope-Tagged Sel-10 in Human Cells, and Perturbation of Amyloid β Peptide Processing by Human Sel-10

Materials and Methods

Construction of Epitope-Tagged Sel-10: Subcloning, Cell Growth and Transfection

An EcoR1 site was introduced in-frame into the human sel-10 cDNA using a polymerase chain reaction (PCR) primed with the oligonucleotides 237 (5′-GGAATTC-CATGAAAAGATTGGACCATGGTTCTG-3′) (SEQ ID NO:18) and 206 (5′-GGA-ATTCCTCACTTCATGT-CACATCAAAGTCCAG-3′) (SEQ ID NO:19). The resulting PCR product was cloned into the EcoR1 site of the vector pCS2+MT. This fused a 5′ 6-myc epitope tag in-frame to the fifth methionine of the hippocampal sel-10 cDNA, i.e., upstream of nucleotide 306 of the sequence given in SEQ ID NO: 1. The nucleotide sequence of this construct, designated 6myc-N-sel-10, is given in SEQ ID NO: 20, while the amino acid sequence of the polypeptide encoded thereby is given in SEQ ID NO: 21. The hippocampal and mammary sel-10 cDNA diverge upstream of this methionine. A PS1 cDNA with a 3′-FLAG tag (PS1-C-FLAG) was subcloned into the pcDNA3.1 vector. An APP cDNA containing the Swedish NL mutation and an attenuated ER retention sequence consisting of the addition of a di-lysyl motif to the C-terminus of APP695 (APP695NL-KK) was cloned into vector pIRES-EGFP (Mullan et al., Nat Genet August 1992; 1(5):345-7). HEK293 and IMR32 cells were grown to 80% confluence in DMEM with 10% FBS and transfected with the above cDNA. A total of 10 mg total DNA/6×10⁶ cells was used for transfection with a single plasmid. For cotransfections of multiple plasmids, an equal amount of each plasmid was used for a total of 10 mg DNA using LipofectAmine (BRL).

In order to construct C-term V5 his tagged sel-10 and the C-term mychis tagged sel-10, the coding sequence of human hippocampal sel-10 was amplified using oligonucleotides primers containing a KpnI restriction site on the 5′ primer: 5′-GGGTA-CCCCTCATTATTCCCTCGAGTTCTTC-3′ (SEQ ID NO:22) and an EcoRI site on the 3′ primer: 5′-GGAATTCCTTCATGTCCACATCAAAGTCC-3′ (SEQ ID NO:23), using the original human sel-10 RACE per product as template. The product was digested with both KpnI and EcoRI and cloned into either the vector pcDNA6/V5-His A or pcDNA3.1/Myc-His(+) A (Invitrogen). The nucleotide sequence of independent isolates was confirmed by dideoxy sequencing. The nucleotide sequence of the C-term V5 his tagged sel-10 is given in SEQ ID NO: 24, while the amino acid sequence of the polypeptide encoded thereby is given in SEQ ID NO: 25. The nucleotide sequence of independent isolates was confirmed by dideoxy sequencing. The nucleotide sequence of the C-term mychis tagged sel-10 is given in SEQ ID NO: 26, while the amino acid sequence of the polypeptide encoded thereby is given in SEQ ID NO: 27.

Clonal Selection of Transformed Cells by FACS

Cell samples were analyzed on an EPICS Elite ESP flow cytometer (Coulter, Hialeah, Fla.) equipped with a 488 nm excitation line supplied by an air-cooled argon laser. EGFP emission was measured through a 525 nm band-pass filter and fluorescence intensity was displayed on a 4-decade log scale after gating on viable cells as determined by forward and right angle light scatter. Single green cells were separated into each well of one 96 well plate containing growth medium without G418. After a four day recovery period, G418 was added to the medium to a final concentration of 400 mg/ml. Wells with clones were expanded from the 96 well plate to a 24 well plate and then to a 6 well plate with the fastest growing colonies chosen for expansion at each passage.

Immunofluorescence

Cells grown on slides were fixed 48 hrs after transfection with 4% formaldehyde and 0.1% Triton X-100 in PBS for 30 min on ice and blocked with 10% Goat serum in PBS (blocking solution) 1 hr RT (i.e., 25° C.), followed by incubation with mouse anti-myc (10 mg/ml) or rabbit anti-FLAG (0.5 mg/ml) antibody 4° C. O/N and then fluorescein-labeled goat anti-mouse or anti-rabbit antibody (5mg/ml) in blocking solution 1 hr at 25° C.

Western Blotting

Cell lysates were made 48 hrs after transfection by incubating 10⁵ cells with 100 ml TENT (50 mM Tris-HCl pH 8.0,2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1× protease inhibitor cocktail) 10 min on ice followed by centrifugation at 14,000 g. The supernatant was loaded on 4-12% NuPage gels (50 mg protein/lane) and electrophoresis and transfer were conducted using an Xcell II Mini-Cell system (Novex). The blot was blocked with 5% milk in PBS 1 hr RT and incubated with anti-myc or anti-FLAG antibody (described in “Immunofluorescence” above) 4° C. O/N, then sheep anti-mouse or anti-rabbit antibody-HRP (0.1 mg/ml) 1 hr RT, followed by Supersignal (Pierce) detection.

ELISA

Cell culture supernatant or cell lysates (100 ml formic acid/10⁶ cells) were assayed in the following double antibody sandwich ELISA, which is capable of detecting levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ peptide in culture supernatant.

Human Aβ 1-40 or 1-42 was measured using monoclonal antibody (mAb) 6E10 (Senetek, St. Louis, Mo.) and biotinylated rabbit antiserum 162 or 164 (NYS Institute for Basic Research, Staten Island, N.Y.) in a double antibody sandwich ELISA. The capture antibody 6E10 is specific to an epitope present on the N-terminal amino acid residues 1-16 of hAβ. The conjugated detecting antibodies 162 and 164 are specific for hAβ 1-40 and 1-42, respectively. The sandwich ELISA was performed according to the method of Pirttila et al. (Neurobiology of Aging 18: 121-7 (1997)). Briefly, a Nunc Maxisorp 96 well immunoplate was coated with 100 μl/well of mAb 6E10 (5 μg/ml) diluted in 0.1M carbonate-bicarbonate buffer, pH 9.6 and incubated at 4° C. overnight. After washing the plate 3× with 0.01M DPBS (Modified Dulbecco's Phosphate Buffered Saline (0.008M sodium phosphate, 0.002M potassium phosphate, 0.14M sodium chloride, 0.01 M potassium chloride, pH 7.4) from Pierce, Rockford, Ill.) containing 0.05% of Tween-20 (DPBST), the plate was blocked for 60 min with 200 μl of 10% normal sheep serum (Sigma) in 0.01M DPBS to avoid non-specific binding. Human Aβ 1-40 or 1-42 standards 100 μl/well (Bachem, Torrance, Calif.) diluted, from a 1 mg/ml stock solution in DMSO, in non transfected conditioned cell medium was added after washing the plate, as well as 100 μl /well of sample i.e. filtered conditioned medium of transfected cells. The plate was incubated for 2 hours at room temperature and 4° C. overnight. The next day, after washing the plate, 100 μl/well biotinylated rabbit antiserum 162 1:400 or 164 1:50 diluted in DPBST+0.5% BSA was added and incubated at room temperature for 1 hr 15 min. Following washes, 100 μl/well neutravidin-horseradish peroxidase (Pierce, Rockford, Ill.) diluted 1:10,000 in DPBST was applied and incubated for 1 hr at room temperature. After the last washes 100 μl well of o-phenylnediamine dihydrochloride (Sigma Chemicals, St. Louis, Mo.) in 50 mM citric acid/100 mM sodium phosphate buffer (Sigma Chemicals, St. Louis, Mo.), pH 5.0, was added as substrate and the color development was monitored at 450 nm in a kinetic microplate reader for 20 min. using Soft max Pro software.

Results

Transfection of HEK293 Cells

Transfection efficiency was monitored through the use of vectors that express green fluorescent protein (GFP) or by immunofluorescent detection of epitope-tagged sel-10 or PS1. An N-terminal 6-myc epitope was used to tag human sel-10 (6 myc-N-sel-10), while PS1 was tagged with a C-terminal FLAG epitope (PS1-C-FLAG). APP695 was modified by inclusion of the Swedish NL mutation to increase Aβ processing and an attenuated endoplasmic reticulum (ER) retention signal consisting of a C-terminal di-lysine motif (APP695NL-KK). The di-lysine motif increases Aβ processing about two fold. The APP695NL-KK construct was inserted into the first cistron of a bicistronic vector containing GFP (pIRES-EGFP, Invitrogen) to allow us to monitor transfection efficiency. Transfection efficiency in HEK293 cells was about 50% for transfections with a single plasmid DNA. For cotransfections with two plasmids, about 30-40% of the cells expressed both proteins as detected by double immunofluorescence.

Expression of recombinant protein in transfected HEK293 cells was confirmed by Western blot as illustrated for PS1-C-FLAG and 6myc-N-sel-10 (FIG. 1A). In the case of cotransfections with three plasmids (PS 1-C-FLAG+6myc-N-sel-10+APP), all three proteins were detected in the same cell lysate by Western blot (FIG. 1B) using appropriate antibodies.

Effect of 6myc-N-sel-10 and PS1-C-FLAG on Aβ Processing

Cotransfection of APP695NL-KK with 6myc-N-sel-10 or PS1-C-FLAG into HEK293 cells increased the release of Ab1-40 and Ab1-42 peptide into the culture supernatant by 2- to 3-fold over transfections with just APP695NL-KK (Table 1). Cotransfection of APP695NL-KK with both 6myc-N-sel-10 and PS1-C-FLAG increased Ab release still further (i.e., 4- to 6-fold increase). In contrast, the ratio of Ab1-42/(Ab1-40+Ab1-42) released into the supernatant decreased about 50%. The subtle decrease in the ratio of Ab1-42/(Ab1-40+Ab1-42) reflects the larger increase in Ab 1-40 relative to Ab 1-42. Neither 6myc-N-sel-10 nor PS1-C-FLAG affected endogenous Ab production in HEK293 cells. Similar observations were also obtained in IMR32 cells (Table 2). However, IMR32 cells transfected less well than HEK293 cells, so the stimulation of APP695NL-KK processing by cotransfection with 6myc-N-sel-10 or PS1-C-FLAG was lower.

Levels of Ab 1-40 expressed in HEK293 cells transfected with APP695NL-KK were sufficient to measure Ab peptide in both the culture supernatant and cell pellet. Considerably more Ab 1-40 is detected in the HEK293 cell pellet than in the supernatant in cells transfected with just APP695NL-KK. Cotransfection with 6myc-N-sel-10 or PS1-C-FLAG proportionately decreased Ab 1-40 in the cell pellet and increased Ab in the culture supernatant. This implies that 6myc-N-sel-10 and PS1-C-FLAG alter processing or trafficking of APP such that proportionately more Ab is released from the cell.

Effect of 6myc-N-sel-10 and PS1-C-FLAG Expression on endogenous Aβ Processing

The effect of 6myc-N-sel-10 on the processing of endogenous APP by human cells was assessed by creating stably transformed HEK293 cell lines expressing these proteins. Two cell lines expressing 6myc-N-sel-10 were derived (sel-10/2 & sel-10/6) as well as a control cell line transformed with pcDNA3.1 vector DNA. Both 6myc-N-sel-10 cell lines expressed the protein as shown by Western blot analysis. Endogenous production of Ab 1-40 was increased in both 6myc-N-sel-10 cell lines in contrast to vector DNA transformed cells Table 3). In addition, stable expression of 6myc-N-sel-10 significantly increased Ab production after transfection with APP695NL-KK plasmid DNA (Table 3). Similar results were obtained with 6 stable cell lines expressing PS1-C-FLAG. All 6 cell lines showed significant elevation of endogenous Aβ processing and all also showed enhanced processing of Ab after transfection with APP695NL-KK (Table 3). In addition, the increase of Aβ processing seen with 6myc-N-sel-10 was also seen with sel-10 tagged at the C-terminus with either mychis or v5his (See Table 4). Both C-terminal and N-terminal tags resulted in an increase in Aβ processing.

Discussion

These data suggest that , when over expressed, 6myc-N-sel-10 as well as PS1-C-FLAG alter Aβ processing in both transient and stable expression systems. A 6-myc epitope tag was used in these experiments to allow detection of sel-10 protein expression by Western blot analysis. If as its sequence homology to yeast CDC4 suggests, sel-10 is an E2-E3 ubiquitin ligase, it should be possible to identify the proteins it targets for ubiquitination. Since the presenilins are degraded via the ubiquitin-proteasome pathway, PS1 & PS2 are logical targets of sel-10 catalyzed ubiquitination [Kim et al., J. Biol. Chem. 272:11006-11010 (1997)]. How sel-10 affects Aβ processing is not understood at this point. In the future, it will be necessary to determine if sel-10 & PS1 increase Aβ processing by altering production, processing, transport, or turn-over of APP, and whether the effect of PS1 is mediated or regulated by sel-10.

These experiments suggest that sel-10 is a potential drug target for decreasing Ab levels in the treatment of AD. They also show that C. elegans is an excellent model system in which to investigate presenilin biology in the context of AD. Thus, as is shown by cotransfection experiments, as well as in stable transformants, expression of 6myc-N-sel10 or PS1-C-FLAG increases Aβ processing. An increase in Aβ processing was seen in both HEK293 cells and IMR32 cells after cotransfection of 6myc-N-sel10 or PS1-C-FLAG with APP695NL-KK. In stable transformants of HEK293 cells expressing 6myc-Sel10 or PS1-C-FLAG, an increase in endogenous Aβ processing was observed, as well as an increase in Aβ processing after transfection with APP695NL-KK. This suggests that inhibitors of either sel-10 and/or PS1, may decrease Aβ processing, and could have therapeutic potential for Alzheimer's disease.

Example 4

SEL-10 Interacts with Presenilin 1, Facilitates its Ubiquitination, and Alters Aβ Production

Mutations in the presenilin genes (PS1 or PS2) in man cause autosomal dominant early onset Alzheimer's disease. These have been linked to alterations in the processing of the amyloid protein precursor (APP)^(1,2). The presenilins are membrane proteins with 6-8 transmembrane domains^(3,4), which localize to the endoplasmic reticulum, Golgi complex, nuclear envelope, kinetochore and centrosome^(5,6). Mutations in sel-12, a Caenorhabditis elegans presenilin cause a defect in egg laying⁷. Sel-12 is one of three nematode presenilin homologous (sel-12, hop-1 and spe-4)⁷⁻⁹. The sel-12 mutant phenotype can be rescued by human PS1 or PS2¹⁰, indicating that PS1, PS2 and sel-12 are functional homologues. Mutations in sel-12 cause a defect in egg laying by altering signaling through the Notch/lin-12 pathway. The sel-12 mutant phenotype can be suppressed by loss of function mutations in a second gene, sel-10¹¹, which probably results in rescue of the egg laying defect by increasing the activity of a functionally redundant presenilin, hop-1^(8,9). SEL-10 is a homologue of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family¹². In this study, we show that human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. This leads to an alteration in the metabolism of APP and to an increase in the production of amyloid β-peptide, the principal component of amyloid plaque in Alzheimer's disease¹³.

The SCF E2-E3 ubiquitin ligases contain a catalytic core consisting of Skp1, Rbx1, Cdc53/CUL-1 and an E2 ubiquitin transferase, Cdc34¹⁴. These are targeted to substrates for ubiquitination by adapter proteins (e.g., Cdc4, Grr1, Met30, β-TrCP) containing an F-box motif and WD40 repeats¹⁵. There is evidence that presenilins are ubiquitinated and undergo degradation through the ubiquitin-proteasome pathway^(16,17). Physical interaction between C. elegans SEL-10 and SEL-12 has been shown previously¹¹. Thus, SEL-10 may be the F-box adaptor protein that recruits presenilins for ubiquitination and subsequent degradation.

Both human and mouse orthologs of C. elegans sel-10 have been identified in EST databases, although the sequence information is incomplete¹². We used rapid amplification of cDNA ends (RACE) to clone amplification products containing the full coding sequences for human and mouse sel-10. There are two variants of human sel-10 cDNA from hippocampus (hippocampus form) and mammary gland (common form) which differ in 5′ sequence upstream of a common translation initiation site. This may indicate that the human sel-10 transcript undergoes differential splicing in a tissue-specific fashion or that the gene contains multiple, tissue specific promoters. The hippocampal form contains four in-frame methionines upstream of the common initiation site and the mammary form contains three. Whether or not these encode proteins with different N-termini is not known. The common transcript is ubiquitously expressed in all tissues tested (FIG. 2A), while the hippocampal form is present only in brain (FIG. 2B). The human sel-10 gene was localized to chromosome 4q31.2-31.3 by in situ hybridization (data not shown). The predicted protein sequences of human and C. elegans SEL-10 have 47.6% amino acid identity and 56.7% similarity. Human SEL-10 contains an F-box domain as found in other SCF family members. It also contains seven WD40/β-transducin repeats¹⁸ as seen in yeast Cdc4p and C. elegans SEL-10, suggesting that the protein forms a seven-bladed propeller structure¹⁹.

We first assessed the physical interaction of human SEL-10 and PS1. SEL-10 tagged with an N-terminal 6-Myc epitope and PS1 were transiently co-expressed in human embryonic kidney cells (HEK293) and their interaction was assessed by immuno-precipitation. Complexes between SEL-10 and PS1 could only be detected in the presence of a proteasome inhibitor, lactacystin, which was added to the cultures at the time of transfection. When immunoprecipitated with anti-PS1 loop antibody, only the immuno-precipitate from cotransfected cells contained SEL-10-myc (FIG. 3A), indicating that SEL-10 can interact with PS1. Since co-immunoprecipitation was not observed in cells without lactacystin treatment (FIG. 3B), this suggests that the complex can only be captured by blocking the entry of PS1 into the proteasome degradation pathway. The interaction between SEL-10 and PS1 was confirmed in the reverse experiment by using anti-myc antibody for immunoprecipitation of SEL-10 (FIG. 3C). PS1 is cleaved within the cytoplasmic loop between transmembrane domains six and seven by an unknown protease which generates N- and C-terminal fragments (PS1-NTF and PS1-CTF, respectively). The SEL-10-myc immunoprecipitates contained primarily full length and high molecular weight forms of PS1, but only very low or undetectable amounts of PS1-NTF and no PS1-CTF. This suggests that SEL-10 may bind primarily to unprocessed PS1.

Next, we examined the effect of SEL-10 co-transfection on PS1 ubiquitination. PS1 immunoprecipitated from cotransfected cells contains a higher level of ubiquitination compared to cells transfected with PS1 alone as shown by probing with anti-ubiquitin antibody (FIG. 4A). This result demonstrates that complex formation between SEL-10 and PS1 facilitates ubiquitination as implied previously by the need for lactacystin to demonstrate SEL-10/PS1 complex accumulation.

We then investigated how ubiquitination affects PS1 protein level. HEK293 cells were cotransfected with either SEL-10 or PS1 alone or in combination and immunoprecipitates were probed with anti-PS1 loop antibody (FIG. 4B). In cells co-transfected with PS1 and SEL-10 in comparison to cells transfected with PS1 alone, PS1-NTF and PS1 -CTF were decreased as were the high molecular weight forms of PS1. However, the amount of unprocessed PS1 appeared to be slightly increased. There are several possible interpretations of this complex cellular effect. The first is that SEL-10 mediated ubiquitination of PS1-CTF and PS1-NTF leads to their degradation. However, the fragments of PS1 are not immunoprecipitated with SEL-10 suggesting that SEL-10 does not directly facilitate ubiquitination of PS1 -NTF and PS1 -CTF. The slight increase in unprocessed PS1 seen in cells cotransfected with SEL-10 may suggest instead that SEL-10 binding to PS1 or SEL-10 mediated ubiquitination of PS1 inhibits proteolytic processing of PS1-NTF and PS1-CTF leading to accumulation of the unprocessed precursor.

In order to examine the impact of SEL-10 mediated ubiquitination of PS1 on amyloid β-peptide (Aβ) production, SEL-10 was expressed by either transient or stable transfection in HEK293 cells with or without wild-type PS1. Aβ peptide production was measured by enzyme immunoassays that could distinguish the 1-40 and 1-42 forms of the peptide. In transient expression experiments (FIG. 5A), coexpression of SEL-10 with APP increased production and release of Aβ1-40 and Aβ1-42 by more than 2-fold in comparison to APP expression alone. Transient coexpression of PS1 with APP also increased Aβ1-40 and Aβ1-42 levels by more than 3-fold, similar to previous reports ²⁰ although this is not a consistent finding². Coexpression of SEL-10 and PS1 with APP had an additive effect with an increase in Aβ production of approximately 7-fold. No effect on the ratio of Aβ1-42/total Aβ was observed with all values falling in the range 8.5% to 13.5%. The stimulation of Aβ processing due to expression of SEL-10 was consistently observed across experiments, however, the degree of stimulation did vary. To confirm and extend the result, a series of HEK293 cell lines were derived with stable expression of SEL-10 or PS1 (FIG. 5B). The two SEL-10 cell lines obtained showed 4- and 7-fold increases in endogenous Aβ1-40 peptide production compared to HEK293 cells transfected with just the pcDNA3.1 vector. Similarly, six cell lines with stable expression of PS1 showed a 2- to 6-fold increase in endogenous Aβ1-40 peptide production. Transient transfection of the stable cell lines with APP cDNA was performed to confirm and extend this result (FIG. 5C). Production of Aβ was greater in the SEL-10 and PS1 cell lines in comparison to a control HEK293 cell line that had been transformed with just the pcDNA3.1 vector. Since SEL-10 decreases the cellular level of PS1-NTF and PS1 -CTF fragments while slightly increasing unprocessed PS1, the increase in unprocessed PS1 may be associated with the increase in Aβ peptide processing observed. Transient expression of PS1 has a similar effect. It increases the cellular level of unprocessed PS1 while having relatively little effect on the levels of PS1-NTF and PS1 -CTF fragments.

Our data indicate that SEL-10 interacts with PS1, stimulates PS1 ubiquitination, and recruits it into the proteasome pathway for protein degradation. SEL-10 is likely to function as an adaptor protein that assembles the core catalytic complex of an SCF E2-E3 ubiquitin ligase¹⁴. Recognition of most SCF substrates by F-box/WD40 repeat adaptor proteins is phosphorylation dependent¹⁵, suggesting that this may be an additional level of cellular regulation of presenilin levels. To demonstrate complex formation between human SEL-10 and PS1 in HEK293 cells required inhibition of proteasome function by lactacystin. In contrast, the C. elegans SEL-10/SEL-12 complex accumulates in human HEK293 cells in the absence of proteasome inhibitors suggesting that nematode SEL-10 is unable to assemble the human core catalytic complex¹¹. Degradation of an eight-pass transmembrane protein such as presenilin presents a topological hurdle since the presenilin protein must be extracted from the membrane and delivered to the proteasome. Like presenilins, a number of other multipass integral membrane proteins with large cytoplasmic domains such as the cystic fibrosis transmembrane conductance regulator (CFTR) are degraded through the ubiquitin-proteasome pathway^(21,22). This pathway also is important for quality control within the endoplasmic reticulum²³ and conceivably could impact on intracellular production of Aβ peptide.

Transfection of PS1 into human cells generally is observed to increase cellular levels of unprocessed PS1, but causes little change in levels of PS1-CTF and PS1-NTF suggesting that processing is under tight cellular control¹⁷, perhaps due to their involvement in apoptotic pathways²⁴. As a consequence of increased transient or stable expression of PS1, we see an increase in Aβ peptide production that probably is due to the accumulation of unprocessed PS1. A similar effect on Aβ peptide production and accumulation of unprocessed PS1 is caused by increased expression of SEL-10. SEL-10 may directly regulate PS1 processing by binding to the cleavage site in the large cytoplasmic loop of PS1, or SEL-10 mediated ubiquitination may block PS1 processing. Similarly, a number of PS1 mutations associated with familial Alzheimer's disease have been shown to decrease processing of PS1 into N- and C-terminal fragments, although that is not a consistent finding²⁵. However, mutant forms of PS1 are associated with a shift in the ratio of Aβ1-42 and Aβ1-40, whereas expression of wild-type PS1 or SEL-10 has no effect on the ratio of Aβ1-42/total Aβ^(1,2).

The genetic data indicates that SEL-10 is a negative regulator of presenilin activity in C. elegans. Loss of SEL-10 function presumably rescues the egg laying defect in sel-12 mutant worms through facilitation of HOP-1 presenilin activity, perhaps by allowing the increased accumulation of processed N- and C-terminal fragments of HOP-1. Sel-10 was identified in a screen for mutations that increase presenilin activity¹¹. In principle, genetic screens in model organisms such as C. elegans or Drosophila can be used to find mutations that decrease presenilin activity, the desired therapeutic goal in Alzheimer's disease²⁶. Such screens have the potential to identify novel therapeutic targets for this devastating disease.

Methods

Cloning

Incyte clone (028971) was identified as the human homologue of C. elegans sel-10 and its sequence was used to design four antisense oligonucleotide primers 5′-TCACT-TCATGTCCACATCAAAGTCC-3′ (SEQ ID NO: 28), 5′-GGTAATTACAAAGTTCTTG-TTGAACTG-3′ (SEQ ID NO: 29), 5′-CCCTGCAACGTGTGTAGACAGG-3′ (SEQ ID NO: 30), and 5′-CCAGTCTCTGCATTCCACACTTTG-3′ (SEQ ID NO: 31), to amplify the remainder of the human sel-10 sequence. “Electronic Northern” analysis revealed expression of sel-10 in hippocampus and mammary gland so these tissues were chosen for 5′RACE cloning using Marathon kit (CloneTech). Marathon-ready cDNA from hippocampus and mammary gland were prepared as directed in the kit. PCR products were cloned into the TA vector pCR3.1 (Invitrogen), and isolates were sequenced. An alternate 5′oligonucleotide primer was also designed based on Incyte clones that have 5′ ends that differ from the hippocampal sel-10 sequence (5′-CTCAGACAGGTCAGGACATTTGG-3′ (SEQ ID NO: 32). Blastn was used to search the Incyte databases LifeSeq and LifeSeqFL. Gap alignments and translations were performed with GCG programs.

Plasmids and Transfections

The human sel-10 cDNA was inserted into the EcoR1 site of the vector pCS2+MT (gift of Jan Kitajewski, Columbia University College of Physicians and Surgeons). This fused a 5′ 6-myc epitope tag in-frame to the fifth methionine of the hippocampal sel-10 cDNA. The hippocampal and mammary sel-10 cDNA diverge upstream of this methionine. A PS1 cDNA with a 3′-FLAG tag (PS1 -C-FLAG) was inserted into the pcDNA3.1 vector. An APP cDNA containing the Swedish KM→NL mutation and an attenuated ER retention sequence consisting of a C-terminal di-lysine motif (APP695Sw-KK) was inserted into the pIRES-EGFP vector (Clontech). HEK293 cells were grown to 80% confluence in DMEM with 10% FBS and transfected with the above cDNAs. A total of 10 μg DNA/6×10⁶ cells was used for transfection with a single plasmid. For cotransfections of multiple plasmids, an equal amount of each plasmid was used for a total of 10 μg DNA using LipofectAmine (BRL). Single cells were sorted into each well of one 96 well plate containing growth medium without G418 by FACS using an EPICS Elite ESP flow cytometer (Coulter, Hialeah, Fla.) equipped with a 488 nm excitation line supplied by an air-cooled argon laser. After a four day recovery period, G418 was added to the medium to a final concentration of 400 μg/ml for the selection of stably transfected cell lines. Stable expression of a cDNA was confirmed by detection of the specific protein or sequence tag by Western blot.

Immunoprecipitation, Western Blot, and ELISA

Aβ and PS1 antibodies have been characterized^(6,27). Cell lysates with equal amount of protein were precipitated with antibody and protein G-Sepharose at 4° C. for 2 hr and beads were washed three times with TENT buffer¹¹. Immunoprecipitates were analyzed by Western blot using 4-12% NuPage Bis-Tris gel and PVDF membrane (Novex), peroxidase-conjugated secondary antibody (Vector) and SuperSignal West Pico Luminol/Enhancer (Pierce). The ELISA for Aβ 1-40 and Aβ 1-42 was performed as described²⁷.

References

1. Borchelt, D. R. et al. Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta 1-42/1-40 ratio in vitro and in vivo. Neuron 17, 1005-1013 (1996).

2. Citron, M. et al. Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. Nat. Med. 3, 67-72 (1997).

3. Doan, A. et al. Protein topology of presenilin 1. Neuron. 17, 1023-1030 (1996).

4. Li, X. & Greenwald, I. Membrane topology of the C. elegans SEL-12 presenilin. Neuron 17, 1015-1021 (1996).

5. De Strooper, B. et al. Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins. J. Biol. Chem. 272, 3590-3598 (1997).

6. Li, J. et al., Alzheimer presenilins in the nuclear membrane, interphase kinetochores, and centrosomes suggest a role in chromosome segregation. Cell 90, 917-927 (1997).

7. Levitan, D. & Greenwald, I. Facilitation of lin, 12, mediated signalling by sel, 12, a Caenorhabditis elegans S182 Alzheimer's disease gene. Nature 377, 351-4 (1995).

8. Li, X. & Greenwald, I. HOP, 1, a Caenorhabditis elegans presenilin, appears to be functionally redundant with SEL, 12 presenilin and to facilitate LIN, 12 and GLP, 1 signaling. Proc. Natl. Acad. Sci. U.S.A. 94,12204-9 (1997).

9. Westlund, B. et al. Reverse genetic analysis of Caenorhabditis elegans presenilins reveals redundant but unequal roles for sel-12 and hop-1 in Notch-pathway signaling. Proc. Natl. Acad. Sci. U.S.A. 96, 2497-2502 (1999).

10. Levitan, D. et al. Assessment of normal and mutant human presenilin function in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 93, 14940-1494 (1996).

11. Wu, G. et al. Evidence for functional and physical association between Caenorhabditis elegans SEL, 10, a Cdc4p, related protein, and SEL, 12 presenilin. Proc. Natl. Acad. Sci. U.S.A. 95, 15787-1 (1998).

12. Hubbard, E. J. et al. Sel, 10, a negative regulator of lin, 12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins. Genes. Dev. 11, 3182-93 (1997).

13. Selkoe, D. J. Physiological production of the b-amyloid protein and the mechanism of Alzheimer's disease. Trends Neurosci. 16, 403-409 (1993).

14. Tyers, M. & Willems, A. R. One ring to rule a superfamily of E3 ubiquitin ligases. Science 284, 601-604 (1999).

15. Patton, E. E. et al., Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. Trends Genet. 14, 236-243 (1998).

16. Steiner, H. et al. Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation. J. Biol. Chem. 273, 32322-32331 (1998).

17. Kim, T. W. et al. Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. J. Biol. Chem. 272, 11006-11010 (1997).

18. Neer E. J. et al. The ancient regulatory-protein family of WD-repeat proteins. Nature 371, 297-300, (1994).

19. Sondek, J. et al. Crystal structure of a G-protein beta gamma dimmer at 2.1A resolution. Nature 379, 369-374 (1996).

20. Ancolio, K. et al. Alpha-secretase-derived product of beta-amyloid precursor protein is decreased by presenilin 1 mutations linked to familial Alzheimer's disease. J. Neurochem. 69, 2494-2499 (1997).

21. Ward, C. L. et al. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83, 121-127 (1995).

22. Johnston, J. A. et al. Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143: 1883-1898 (1998).

23. Kopito, R. R. ER quality control: the cytoplasmic connection. Cell. 88, 427-430 (1997).

24. Zhang, Z. et al. Destabilization of beta-catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature. 395: 698-702 (1998).

25. Shirotani, K. et al. Effects of presenilin N-terminal fragments on production of amyloid b peptide and accumulation of endogenous presenilins. Neurosci. Let. 262, 37-40 (1999).

26. De Strooper, B. et al. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387-390 (1998).

27. Mehta, P. D. et al. Increased plasma amyloid beta protein 1-42 levels in Down syndrome. Neurosci Lett 241, 13-16 (1998).

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention.

The entire disclosure of all publications cited herein are hereby incorporated by reference.

TABLE 1 Effect of 6myc-N-sel-10 and PS1-C-FLAG transient transfection on Ab levels in HEK293 cell supernatants. Plasmids Ab1-42 Ab1-40 Ab1-42/total Ab Transfected ng/ml ng/ml ng/ml pcDNA3  81 ± 20 231 ± 50 0.26 ± 0.05 6myc-N-sel-10  67 ± 7 246 ± 34 0.21 ± 0.03 PS1-C-FLAG  75 ± 18 227 ± 45 0.25 ± 0.03 PS1-C-FLAG +  77 ± 21 220 ± 26 0.25 ± 0.03 6myc-N-sel-10 APP695NL-KK 141 ± 27  896 ± 103 0.14 ± 0.02 APP695NL-KK + 308 ± 17 2576 ± 190 0.11 ± 0.00 6myc-N-sel-10 APP695NL-KK + 364 ± 39 3334 ± 337 0.09 ± 0.00 PS1-C-FLAG APP695NL-KK + 550 ± 20 5897 ± 388 0.09 ± 0.00 PS1-C-FLAG + 6myc-N-sel-10

TABLE 2 Effect of 6myc-N-sel-10 and PS1-C-FLAG transient transfected on Ab levels in IMR32 cell supernatants. Plasmids Ab1-42 Ab1-40 Ab1-42/total Ab Transfected ng/ml ng/ml ng/ml pcDNA3 65 ± 3 319 ± 146 0.19 ± 0.06 6myc-N-sel-10 63 ± 0 246 ± 53 0.21 ± 0.04 PS1-C-FLAG 67 ± 6 307 ± 79 0.18 ± 0.04 PS1-C-FLAG + 67 ± 6 302 ± 94 0.20 ± 0.08 6myc-N-sel-10 APP695NL-KK 66 ± 5 348 ± 110 0.17 ± 0.05 APP695NL-KK + 75 ± 18 448 ± 141 0.15 ± 0.03 6myc-N-sel-10 APP695NL-KK + 63 ± 26 446 ± 72 0.12 ± 0.02 PS1-C-FLAG APP695NL-KK + 81 ± 26 565 ± 179 0.12 ± 0.01 PS1-C-FLAG + 6myc-N-sel-10

TABLE 3 Endogenous and exogenous Ab1-40 and Ab1-42 levels in supernatants from stable transformants of HEK293 cells. GFP APP695NL-KK Transfection Transfection Ab1-40 Ab1-42 Ab1-40 Ab1-42 Stable Line ng/ml ng/ml ng/ml ng/ml 6myc-N-sel10/2 297 ± 29 109 ± 17  4877 ± 547 750 ± 32 6myc-N-sel10/6 168 ± 18 85 ± 11 8310 ± 308 1913 ± 19  PS1-C-FLAG/2  97 ± 6 68 ± 8  3348 ± 68 493 ± 21 PS1-C-FLAG/8 118 ± 11 85 ± 17 3516 ± 364 515 ± 36 PS1-C-FLAG/9  83 ± 20 67 ± 16 2369 ± 73 350 ± 12 PS1-C-FLAG/11 152 ± 17 68 ± 13 4771 ± 325 599 ± 25 PS1-C-FLAG/12 141 ± 12 50 ± 10 4095 ± 210 449 ± 21 PS1-C-FLAG/13 270 ± 139 61 ± 28 6983 ± 304 745 ± 41 pcDNA3/1  43 ± 13 75 ± 15 1960 ± 234 61 ± 6

TABLE 4 Sel-10 constructs with epitope tags at the N or C terminus increase Aβ 1-40 and Aβ 1-42. construct Aβ 1-40 % increase P-value Aβ 1-42 % increase P-value pcDNA 4240 ± 102 614 ± 10 6myc-N-sel-10 7631 ± 465 80% 3.7 × 10⁻⁶ 1136 ± 73  46% 7.9 × 10⁻⁶ sel-10-C-mychis 5485 ± 329 29% 1.8 × 10⁻⁴ 795 ± 50 29% 4.0 × 10⁻⁴ sel-10-C-V5his 6210 ± 498 46% 1.2 × 10⁻⁴ 906 ± 73 48% 2.1 × 10⁻⁴

32 1 3550 DNA Homo sapiens misc_feature (2485)..(2485) residue uncertain 1 ctcattattc cctcgagttc ttctcagtca agctgcatgt atgtatgtgt gtcccgagaa 60 gcggtttgat actgagctgc atttgccttt actgtggagt tttgttgccg gttctgctcc 120 ctaatcttcc ttttctgacg tgcctgagca tgtccacatt agaatctgtg acatacctac 180 ctgaaaaagg tttatattgt cagagactgc caagcagccg gacacacggg ggcacagaat 240 cactgaaggg gaaaaataca gaaaatatgg gtttctacgg cacattaaaa atgatttttt 300 acaaaatgaa aagaaagttg gaccatggtt ctgaggtccg ctctttttct ttgggaaaga 360 aaccatgcaa agtctcagaa tatacaagta ccactgggct tgtaccatgt tcagcaacac 420 caacaacttt tggggacctc agagcagcca atggccaagg gcaacaacga cgccgaatta 480 catctgtcca gccacctaca ggcctccagg aatggctaaa aatgtttcag agctggagtg 540 gaccagagaa attgcttgct ttagatgaac tcattgatag ttgtgaacca acacaagtaa 600 aacatatgat gcaagtgata gaaccccagt ttcaacgaga cttcatttca ttgctcccta 660 aagagttggc actctatgtg ctttcattcc tggaacccaa agacctgcta caagcagctc 720 agacatgtcg ctactggaga attttggctg aagacaacct tctctggaga gagaaatgca 780 aagaagaggg gattgatgaa ccattgcaca tcaagagaag aaaagtaata aaaccaggtt 840 tcatacacag tccatggaaa agtgcataca tcagacagca cagaattgat actaactgga 900 ggcgaggaga actcaaatct cctaaggtgc tgaaaggaca tgatgatcat gtgatcacat 960 gcttacagtt ttgtggtaac cgaatagtta gtggttctga tgacaacact ttaaaagttt 1020 ggtcagcagt cacaggcaaa tgtctgagaa cattagtggg acatacaggt ggagtatggt 1080 catcacaaat gagagacaac atcatcatta gtggatctac agatcggaca ctcaaagtgt 1140 ggaatgcaga gactggagaa tgtatacaca ccttatatgg gcatacttcc actgtgcgtt 1200 gtatgcatct tcatgaaaaa agagttgtta gcggttctcg agatgccact cttagggttt 1260 gggatattga gacaggccag tgtttacatg ttttgatggg tcatgttgca gcagtccgct 1320 gtgttcaata tgatggcagg agggttgtta gtggagcata tgattttatg gtaaaggtgt 1380 gggatccaga gactgaaacc tgtctacaca cgttgcaggg gcatactaat agagtctatt 1440 cattacagtt tgatggtatc catgtggtga gtggatctct tgatacatca atccgtgttt 1500 gggatgtgga gacagggaat tgcattcaca cgttaacagg gcaccagtcg ttaacaagtg 1560 gaatggaact caaagacaat attcttgtct ctgggaatgc agattctaca gttaaaatct 1620 gggatatcaa aacaggacag tgtttacaaa cattgcaagg tcccaacaag catcagagtg 1680 ctgtgacctg tttacagttc aacaagaact ttgtaattac cagctcagat gatggaactg 1740 taaaactatg ggacttgaaa acgggtgaat ttattcgaaa cctagtcaca ttggagagtg 1800 gggggagtgg gggagttgtg tggcggatca gagcctcaaa cacaaagctg gtgtgtgcag 1860 ttgggagtcg gaatgggact gaagaaacca agctgctggt gctggacttt gatgtggaca 1920 tgaagtgaag agcagaaaag atgaatttgt ccaattgtgt agacgatata ctccctgccc 1980 ttccccctgc aaaaagaaaa aaagaaaaga aaaagaaaaa aatcccttgt tctcagtggt 2040 gcaggatgtt ggcttggggc aacagattga aaagacctac agactaagaa ggaaaagaag 2100 aagagatgac aaaccataac tgacaagaga ggcgtctgct gtctcatcac ataaaaggct 2160 tcacttttga ctgagggcag ctttgcaaaa tgagactttc taaatcaaac caggtgcaat 2220 tatttcttta ttttcttctc cagtggtcat tggggcagtg ttaatgctga aacatcatta 2280 cagattctgc tagcctgttc ttttaccact gacagctaga cacctagaaa ggaactgcaa 2340 taatatcaaa acaagtactg gttgactttc taattagaga gcatctgcaa caaaaagtca 2400 tttttctgga gtggaaaagc ttaaaaaaat tactgtgaat tgtttttgta cagttatcat 2460 gaaaagcttt tttttttatt ttttngccaa ccattgccaa tgtcaatcaa tcacagtatt 2520 agcctctgtt aatctattta ctgttgcttc catatacatt cttcaatgca tatgttgctc 2580 aaaggtggca agttgtcctg ggttctgtga gtcctgagat ggatttaatt cttgatgctg 2640 gtgctagaag taggtcttca aatatgggat tgttgtccca accctgtact gtactcccag 2700 tggccaaact tatttatgct gctaaatgaa agaaagaaaa aagcaaatta ttttttttat 2760 tttttttctg ctgtgacgtt ttagtcccag actgaattcc aaatttgctc tagtttggtt 2820 atggaaaaaa gactttttgc cactgaaact tgagccatct gtgcctctaa gaggctgaga 2880 atggaagagt ttcagataat aaagagtgaa gtttgcctgc aagtaaagaa ttgagagtgt 2940 gtgcaaagct tattttcttt tatctgggca aaaattaaaa cacattcctt ggaacagagc 3000 tattacttgc ctgttctgtg gagaaacttt tctttttgag ggctgtggtg aatggatgaa 3060 cgtacatcgt aaaactgaca aaatatttta aaaatatata aaacacaaaa ttaaaataaa 3120 gttgctggtc agtcttagtg ttttacagta tttgggaaaa caactgttac agttttattg 3180 ctctgagtaa ctgacaaagc agaaactatt cagtttttgt agtaaaggcg tcacatgcaa 3240 acaaacaaaa tgaatgaaac agtcaaatgg tttgcctcat tctccaagag ccacaactca 3300 agctgaactg tgaaagtggt ttaacactgt atcctaggcg atcttttttc ctccttctgt 3360 ttattttttt gnttgtttta tttatagtct gatttaaaac aatcagattc aagttggtta 3420 attttagtta tgtaacaacc tgacatgatg gaggaaaaca acctttaaag ggattgtgtc 3480 tatggtttga ttcacttaga aattttattt tcttataact taagtgcaat aaaatgtgtt 3540 ttttcatgtt 3550 2 3571 DNA Homo sapiens misc_feature (2506)..(2506) residue uncertain 2 ctcagcaggt caggacattt ggtaggggaa ggttgaaaga caaaagcagc aggccttggg 60 ttctcagcct tttaaaaact attattaaat atatattttt aaaatttagt ggttagagct 120 tttagtaatg tgcctgtatt acatgtagag agtattcgtc aaccaagagg agttttaaaa 180 tgtcaaaacc gggaaaacct actctaaacc atggcttggt tcctgttgat cttaaaagtg 240 caaaagagcc tctaccacat caaaccgtga tgaagatatt tagcattagc atcattgccc 300 aaggcctccc tttttgtcga agacggatga aaagaaagtt ggaccatggt tctgaggtcc 360 gctctttttc tttgggaaag aaaccatgca aagtctcaga atatacaagt accactgggc 420 ttgtaccatg ttcagcaaca ccaacaactt ttggggacct cagagcagcc aatggccaag 480 ggcaacaacg acgccgaatt acatctgtcc agccacctac aggcctccag gaatggctaa 540 aaatgtttca gagctggagt ggaccagaga aattgcttgc tttagatgaa ctcattgata 600 gttgtgaacc aacacaagta aaacatatga tgcaagtgat agaaccccag tttcaacgag 660 acttcatttc attgctccct aaagagttgg cactctatgt gctttcattc ctggaaccca 720 aagacctgct acaagcagct cagacatgtc gctactggag aattttggct gaagacaacc 780 ttctctggag agagaaatgc aaagaagagg ggattgatga accattgcac atcaagagaa 840 gaaaagtaat aaaaccaggt ttcatacaca gtccatggaa aagtgcatac atcagacagc 900 acagaattga tactaactgg aggcgaggag aactcaaatc tcctaaggtg ctgaaaggac 960 atgatgatca tgtgatcaca tgcttacagt tttgtggtaa ccgaatagtt agtggttctg 1020 atgacaacac tttaaaagtt tggtcagcag tcacaggcaa atgtctgaga acattagtgg 1080 gacatacagg tggagtatgg tcatcacaaa tgagagacaa catcatcatt agtggatcta 1140 cagatcggac actcaaagtg tggaatgcag agactggaga atgtatacac accttatatg 1200 ggcatacttc cactgtgcgt tgtatgcatc ttcatgaaaa aagagttgtt agcggttctc 1260 gagatgccac tcttagggtt tgggatattg agacaggcca gtgtttacat gttttgatgg 1320 gtcatgttgc agcagtccgc tgtgttcaat atgatggcag gagggttgtt agtggagcat 1380 atgattttat ggtaaaggtg tgggatccag agactgaaac ctgtctacac acgttgcagg 1440 ggcatactaa tagagtctat tcattacagt ttgatggtat ccatgtggtg agtggatctc 1500 ttgatacatc aatccgtgtt tgggatgtgg agacagggaa ttgcattcac acgttaacag 1560 ggcaccagtc gttaacaagt ggaatggaac tcaaagacaa tattcttgtc tctgggaatg 1620 cagattctac agttaaaatc tgggatatca aaacaggaca gtgtttacaa acattgcaag 1680 gtcccaacaa gcatcagagt gctgtgacct gtttacagtt caacaagaac tttgtaatta 1740 ccagctcaga tgatggaact gtaaaactat gggacttgaa aacgggtgaa tttattcgaa 1800 acctagtcac attggagagt ggggggagtg ggggagttgt gtggcggatc agagcctcaa 1860 acacaaagct ggtgtgtgca gttgggagtc ggaatgggac tgaagaaacc aagctgctgg 1920 tgctggactt tgatgtggac atgaagtgaa gagcagaaaa gatgaatttg tccaattgtg 1980 tagacgatat actccctgcc cttccccctg caaaaagaaa aaaagaaaag aaaaagaaaa 2040 aaatcccttg ttctcagtgg tgcaggatgt tggcttgggg caacagattg aaaagaccta 2100 cagactaaga aggaaaagaa gaagagatga caaaccataa ctgacaagag aggcgtctgc 2160 tgtctcatca cataaaaggc ttcacttttg actgagggca gctttgcaaa atgagacttt 2220 ctaaatcaaa ccaggtgcaa ttatttcttt attttcttct ccagtggtca ttggggcagt 2280 gttaatgctg aaacatcatt acagattctg ctagcctgtt cttttaccac tgacagctag 2340 acacctagaa aggaactgca ataatatcaa aacaagtact ggttgacttt ctaattagag 2400 agcatctgca acaaaaagtc atttttctgg agtggaaaag cttaaaaaaa ttactgtgaa 2460 ttgtttttgt acagttatca tgaaaagctt ttttttttat tttttngcca accattgcca 2520 atgtcaatca atcacagtat tagcctctgt taatctattt actgttgctt ccatatacat 2580 tcttcaatgc atatgttgct caaaggtggc aagttgtcct gggttctgtg agtcctgaga 2640 tggatttaat tcttgatgct ggtgctagaa gtaggtcttc aaatatggga ttgttgtccc 2700 aaccctgtac tgtactccca gtggccaaac ttatttatgc tgctaaatga aagaaagaaa 2760 aaagcaaatt atttttttta ttttttttct gctgtgacgt tttagtccca gactgaattc 2820 caaatttgct ctagtttggt tatggaaaaa agactttttg ccactgaaac ttgagccatc 2880 tgtgcctcta agaggctgag aatggaagag tttcagataa taaagagtga agtttgcctg 2940 caagtaaaga attgagagtg tgtgcaaagc ttattttctt ttatctgggc aaaaattaaa 3000 acacattcct tggaacagag ctattacttg cctgttctgt ggagaaactt ttctttttga 3060 gggctgtggt gaatggatga acgtacatcg taaaactgac aaaatatttt aaaaatatat 3120 aaaacacaaa attaaaataa agttgctggt cagtcttagt gttttacagt atttgggaaa 3180 acaactgtta cagttttatt gctctgagta actgacaaag cagaaactat tcagtttttg 3240 tagtaaaggc gtcacatgca aacaaacaaa atgaatgaaa cagtcaaatg gtttgcctca 3300 ttctccaaga gccacaactc aagctgaact gtgaaagtgg tttaacactg tatcctaggc 3360 gatctttttt cctccttctg tttatttttt tgnttgtttt atttatagtc tgatttaaaa 3420 caatcagatt caagttggtt aattttagtt atgtaacaac ctgacatgat ggaggaaaac 3480 aacctttaaa gggattgtgt ctatggtttg attcacttag aaattttatt ttcttataac 3540 ttaagtgcaa taaaatgtgt tttttcatgt t 3571 3 627 PRT Homo sapiens 3 Met Cys Val Pro Arg Ser Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly Val Leu Leu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys Leu Ser Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40 45 Gly Leu Tyr Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr 50 55 60 Glu Ser Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr 65 70 75 80 Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser 85 90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135 140 Ile Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145 150 155 160 Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu 165 170 175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile 180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile 290 295 300 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp 305 310 315 320 Asn Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr 325 330 335 Leu Val Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn 340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val 370 375 380 Arg Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385 390 395 400 Ala Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val 405 410 415 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg 420 425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln 530 535 540 Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser 545 550 555 560 Ser Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe 565 570 575 Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val 580 585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val 610 615 620 Asp Met Lys 625 4 592 PRT Homo sapiens 4 Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys Gly Leu Tyr 1 5 10 15 Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr Glu Ser Leu 20 25 30 Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr Leu Lys Met 35 40 45 Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg 50 55 60 Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser 65 70 75 80 Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp 85 90 95 Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser 100 105 110 Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser 115 120 125 Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser 130 135 140 Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln 145 150 155 160 Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr 165 170 175 Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr 180 185 190 Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu 195 200 205 Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg 210 215 220 Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr 225 230 235 240 Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys 245 250 255 Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu 260 265 270 Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu 275 280 285 Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly 290 295 300 His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile 305 310 315 320 Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly 325 330 335 Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met 340 345 350 His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu 355 360 365 Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly 370 375 380 His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val 385 390 395 400 Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu 405 410 415 Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu 420 425 430 Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile 435 440 445 Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly 450 455 460 His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val 465 470 475 480 Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly 485 490 495 Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val 500 505 510 Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp 515 520 525 Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn 530 535 540 Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg Ile 545 550 555 560 Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly 565 570 575 Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 580 585 590 5 553 PRT Homo sapiens 5 Met Gly Phe Tyr Gly Thr Leu Lys Met Ile Phe Tyr Lys Met Lys Arg 1 5 10 15 Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser Leu Gly Lys Lys 20 25 30 Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys 35 40 45 Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln 50 55 60 Gly Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro Pro Thr Gly Leu 65 70 75 80 Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu 85 90 95 Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro Thr Gln Val Lys 100 105 110 His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser 115 120 125 Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro 130 135 140 Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu 145 150 155 160 Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys Glu Glu Gly Ile 165 170 175 Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val Ile Lys Pro Gly Phe 180 185 190 Ile His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln His Arg Ile Asp 195 200 205 Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val Leu Lys Gly 210 215 220 His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile 225 230 235 240 Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val Trp Ser Ala Val Thr 245 250 255 Gly Lys Cys Leu Arg Thr Leu Val Gly His Thr Gly Gly Val Trp Ser 260 265 270 Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr 275 280 285 Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His Thr Leu Tyr 290 295 300 Gly His Thr Ser Thr Val Arg Cys Met His Leu His Glu Lys Arg Val 305 310 315 320 Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp Asp Ile Glu Thr 325 330 335 Gly Gln Cys Leu His Val Leu Met Gly His Val Ala Ala Val Arg Cys 340 345 350 Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala Tyr Asp Phe Met 355 360 365 Val Lys Val Trp Asp Pro Glu Thr Glu Thr Cys Leu His Thr Leu Gln 370 375 380 Gly His Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp Gly Ile His Val 385 390 395 400 Val Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp Asp Val Glu Thr 405 410 415 Gly Asn Cys Ile His Thr Leu Thr Gly His Gln Ser Leu Thr Ser Gly 420 425 430 Met Glu Leu Lys Asp Asn Ile Leu Val Ser Gly Asn Ala Asp Ser Thr 435 440 445 Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln 450 455 460 Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu Gln Phe Asn Lys 465 470 475 480 Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val Lys Leu Trp Asp 485 490 495 Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr Leu Glu Ser Gly 500 505 510 Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu 515 520 525 Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu 530 535 540 Val Leu Asp Phe Asp Val Asp Met Lys 545 550 6 545 PRT Homo sapiens 6 Met Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser Glu Val 1 5 10 15 Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr 20 25 30 Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly 35 40 45 Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr 50 55 60 Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln 65 70 75 80 Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp 85 90 95 Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro 100 105 110 Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu 115 120 125 Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln 130 135 140 Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg 145 150 155 160 Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg 165 170 175 Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala 180 185 190 Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu 195 200 205 Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile Thr Cys 210 215 220 Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr 225 230 235 240 Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val 245 250 255 Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile 260 265 270 Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr 275 280 285 Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys 290 295 300 Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr 305 310 315 320 Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met 325 330 335 Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val 340 345 350 Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr 355 360 365 Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser 370 375 380 Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser 385 390 395 400 Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr 405 410 415 Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu 420 425 430 Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr 435 440 445 Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala 450 455 460 Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp 465 470 475 480 Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg 485 490 495 Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg 500 505 510 Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn 515 520 525 Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met 530 535 540 Lys 545 7 540 PRT Homo sapiens 7 Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser Leu 1 5 10 15 Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly Leu 20 25 30 Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala Ala 35 40 45 Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro Pro 50 55 60 Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly Pro 65 70 75 80 Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro Thr 85 90 95 Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg Asp 100 105 110 Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu Ser Phe 115 120 125 Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr Trp 130 135 140 Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys Glu 145 150 155 160 Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val Ile Lys 165 170 175 Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln His 180 185 190 Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val 195 200 205 Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys Gly 210 215 220 Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val Trp Ser 225 230 235 240 Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly His Thr Gly Gly 245 250 255 Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser Thr 260 265 270 Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His 275 280 285 Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His Leu His Glu 290 295 300 Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp Asp 305 310 315 320 Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His Val Ala Ala 325 330 335 Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala Tyr 340 345 350 Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr Cys Leu His 355 360 365 Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp Gly 370 375 380 Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp Asp 385 390 395 400 Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His Gln Ser Leu 405 410 415 Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser Gly Asn Ala 420 425 430 Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln 435 440 445 Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu Gln 450 455 460 Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val Lys 465 470 475 480 Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr Leu 485 490 495 Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser Asn 500 505 510 Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu Thr 515 520 525 Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 530 535 540 8 589 PRT Homo sapiens 8 Met Ser Lys Pro Gly Lys Pro Thr Leu Asn His Gly Leu Val Pro Val 1 5 10 15 Asp Leu Lys Ser Ala Lys Glu Pro Leu Pro His Gln Thr Val Met Lys 20 25 30 Ile Phe Ser Ile Ser Ile Ile Ala Gln Gly Leu Pro Phe Cys Arg Arg 35 40 45 Arg Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser 50 55 60 Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly 65 70 75 80 Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala 85 90 95 Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro 100 105 110 Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly 115 120 125 Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro 130 135 140 Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg 145 150 155 160 Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu Ser 165 170 175 Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr 180 185 190 Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys 195 200 205 Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val Ile 210 215 220 Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln 225 230 235 240 His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys 245 250 255 Val Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys 260 265 270 Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val Trp 275 280 285 Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly His Thr Gly 290 295 300 Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser 305 310 315 320 Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile 325 330 335 His Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His Leu His 340 345 350 Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp 355 360 365 Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His Val Ala 370 375 380 Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala 385 390 395 400 Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr Cys Leu 405 410 415 His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp 420 425 430 Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp 435 440 445 Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His Gln Ser 450 455 460 Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser Gly Asn 465 470 475 480 Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu 485 490 495 Gln Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu 500 505 510 Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val 515 520 525 Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr 530 535 540 Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser 545 550 555 560 Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu 565 570 575 Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 580 585 9 559 PRT Homo sapiens 9 Met Lys Ile Phe Ser Ile Ser Ile Ile Ala Gln Gly Leu Pro Phe Cys 1 5 10 15 Arg Arg Arg Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser 20 25 30 Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr 35 40 45 Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu 50 55 60 Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser Val 65 70 75 80 Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp 85 90 95 Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys 100 105 110 Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln Phe 115 120 125 Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val 130 135 140 Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys 145 150 155 160 Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys 165 170 175 Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys 180 185 190 Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr Ile 195 200 205 Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser 210 215 220 Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln 225 230 235 240 Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys 245 250 255 Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly His 260 265 270 Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile Ser 275 280 285 Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu 290 295 300 Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His 305 310 315 320 Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg 325 330 335 Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His 340 345 350 Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser 355 360 365 Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr 370 375 380 Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu Gln 385 390 395 400 Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile Arg 405 410 415 Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His 420 425 430 Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser 435 440 445 Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln 450 455 460 Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr 465 470 475 480 Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly 485 490 495 Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu 500 505 510 Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg Ile Arg 515 520 525 Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr 530 535 540 Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 545 550 555 10 540 PRT Homo sapiens 10 Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser Leu 1 5 10 15 Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly Leu 20 25 30 Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala Ala 35 40 45 Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro Pro 50 55 60 Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly Pro 65 70 75 80 Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro Thr 85 90 95 Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg Asp 100 105 110 Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu Ser Phe 115 120 125 Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr Trp 130 135 140 Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys Glu 145 150 155 160 Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val Ile Lys 165 170 175 Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln His 180 185 190 Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val 195 200 205 Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys Gly 210 215 220 Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val Trp Ser 225 230 235 240 Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly His Thr Gly Gly 245 250 255 Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser Thr 260 265 270 Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His 275 280 285 Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His Leu His Glu 290 295 300 Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp Asp 305 310 315 320 Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His Val Ala Ala 325 330 335 Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala Tyr 340 345 350 Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr Cys Leu His 355 360 365 Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp Gly 370 375 380 Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp Asp 385 390 395 400 Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His Gln Ser Leu 405 410 415 Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser Gly Asn Ala 420 425 430 Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln 435 440 445 Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu Gln 450 455 460 Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val Lys 465 470 475 480 Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr Leu 485 490 495 Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser Asn 500 505 510 Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu Thr 515 520 525 Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 530 535 540 11 34 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 11 cgggatccac catggatgat ggatcgatga cacc 34 12 33 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 12 ggaattcctt aagggtatac agcatcaaag tcg 33 13 25 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 13 tcacttcatg tccacatcaa agtcc 25 14 26 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 14 ggtaattaca agttcttgtt gaactg 26 15 22 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 15 ccctgcaacg tgtgtagaca gg 22 16 24 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 16 ccagtctctg cattccacac tttg 24 17 23 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 17 ctcagacagg tcaggacatt tgg 23 18 33 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 18 ggaattccat gaaaagattg gaccatggtt ctg 33 19 34 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 19 ggaattcctc acttcatgtc acatcaaagt ccag 34 20 1881 DNA Artificial Sequence Description of Artificial Sequence 6 myc tagged homo sapiens 20 atggagcaaa agctcatttc tgaagaggac ttgaatgaaa tggagcaaaa gctcatttct 60 gaagaggact tgaatgaaat ggagcaaaag ctcatttctg aagaggactt gaatgaaatg 120 gagcaaaagc tcatttctga agaggacttg aatgaaatgg agcaaaagct catttctgaa 180 gaggacttga atgaaatgga gagcttgggc gacctcacca tggagcaaaa gctcatttct 240 gaagaggact tgaattccat gaaaagaaag ttggaccatg gttctgaggt ccgctctttt 300 tctttgggaa agaaaccatg caaagtctca gaatatacaa gtaccactgg gcttgtacca 360 tgttcagcaa caccaacaac ttttggggac ctcagagcag ccaatggcca agggcaacaa 420 cgacgccgaa ttacatctgt ccagccacct acaggcctcc aggaatggct aaaaatgttt 480 cagagctgga gtggaccaga gaaattgctt gctttagatg aactcattga tagttgtgaa 540 ccaacacaag taaaacatat gatgcaagtg atagaacccc agtttcaacg agacttcatt 600 tcattgctcc ctaaagagtt ggcactctat gtgctttcat tcctggaacc caaagacctg 660 ctacaagcag ctcagacatg tcgctactgg agaattttgg ctgaagacaa ccttctctgg 720 agagagaaat gcaaagaaga ggggattgat gaaccattgc acatcaagag aagaaaagta 780 ataaaaccag gtttcataca cagtccatgg aaaagtgcat acatcagaca gcacagaatt 840 gatactaact ggaggcgagg agaactcaaa tctcctaagg tgctgaaagg acatgatgat 900 catgtgatca catgcttaca gttttgtggt aaccgaatag ttagtggttc tgatgacaac 960 actttaaaag tttggtcagc agtcacaggc aaatgtctga gaacattagt gggacataca 1020 ggtggagtat ggtcatcaca aatgagggac aacatcatca ttagtggatc tacagatcgg 1080 acactcaaag tgtggaatgc agagactgga gaatgtatac acaccttata tgggcatact 1140 tccactgtgc gttgtatgca tcttcatgaa aaaagagttg ttagcggttc tcgagatgcc 1200 actcttaggg tttgggatat tgagacaggc cagtgtttac atgttttgat gggtcatgtt 1260 gcagcagtcc gctgtgttca atatgatggc aggagggttg ttagtggagc atatgatttt 1320 atggtaaagg tgtgggatcc agagactgaa acctgtctac acacgttgca ggggcatact 1380 aatagagtct attcattaca gtttgatggt atccatgtgg tgagtggatc tcttgataca 1440 tccatccgtg tttgggatgt ggagacaggg aattgcattc acacgttaac agggcaccag 1500 tcgttaacaa gtggaatgga actcaaagac aatattcttg tctctgggaa tgcagattct 1560 acagttaaaa tctgggatat caaaacagga cagtgtttac aaacattgca aggtcccaac 1620 aagcatcaga gtgctgtgac ctgtttacag ttcaacaaga actttgtaat taccagctca 1680 gatgatggaa ctgtaaaact atgggacttg aaaacgggtg aatttattcg aaacctagtc 1740 acattggaga gtggggggag tgggggagtt gtgtggcgga tcagagcctc aaacacaaag 1800 ctggtgtgtg cagttgggag tcggaatggg actgaagaaa ccaagctgct ggtgctggac 1860 tttgatgtgg acatgaagtg a 1881 21 626 PRT Artificial Sequence Description of Artificial Sequence 6 myc tagged homo sapien 21 Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Glu Met Glu Gln 1 5 10 15 Lys Leu Ile Ser Glu Glu Asp Leu Asn Glu Met Glu Gln Lys Leu Ile 20 25 30 Ser Glu Glu Asp Leu Asn Glu Met Glu Gln Lys Leu Ile Ser Glu Glu 35 40 45 Asp Leu Asn Glu Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 50 55 60 Glu Met Glu Ser Leu Gly Asp Leu Thr Met Glu Gln Lys Leu Ile Ser 65 70 75 80 Glu Glu Asp Leu Asn Ser Met Lys Arg Lys Leu Asp His Gly Ser Glu 85 90 95 Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr 100 105 110 Thr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe 115 120 125 Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile 130 135 140 Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe 145 150 155 160 Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile 165 170 175 Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu 180 185 190 Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala 195 200 205 Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala 210 215 220 Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp 225 230 235 240 Arg Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys 245 250 255 Arg Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser 260 265 270 Ala Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu 275 280 285 Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile Thr 290 295 300 Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn 305 310 315 320 Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu 325 330 335 Val Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile 340 345 350 Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu 355 360 365 Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg 370 375 380 Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala 385 390 395 400 Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu 405 410 415 Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg 420 425 430 Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu 435 440 445 Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr 450 455 460 Ser Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp Thr 465 470 475 480 Ser Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu 485 490 495 Thr Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile 500 505 510 Leu Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys 515 520 525 Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln Ser 530 535 540 Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser 545 550 555 560 Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile 565 570 575 Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp 580 585 590 Arg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg 595 600 605 Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp 610 615 620 Met Lys 625 22 31 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 22 gggtacccct cattattccc tcgagttctt c 31 23 29 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 23 ggaattcctt catgtccaca tcaaagtcc 29 24 2010 DNA Artificial Sequence Description of Artificial Sequence V5HIS tagged homo sapien 24 atgtgtgtcc cgagaagcgg tttgatactg agctgcattt gcctttactg tggagttttg 60 ttgccggttc tgctccctaa tcttcctttt ctgacgtgcc tgagcatgtc cacattagaa 120 tctgtgacat acctacctga aaaaggttta tattgtcaga gactgccaag cagccggaca 180 cacgggggca cagaatcact gaaggggaaa aatacagaaa atatgggttt ctacggcaca 240 ttaaaaatga ttttttacaa aatgaaaaga aagttggacc atggttctga ggtccgctct 300 ttttctttgg gaaagaaacc atgcaaagtc tcagaatata caagtaccac tgggcttgta 360 ccatgttcag caacaccaac aacttttggg gacctcagag cagccaatgg ccaagggcaa 420 caacgacgcc gaattacatc tgtccagcca cctacaggcc tccaggaatg gctaaaaatg 480 tttcagagct ggagtggacc agagaaattg cttgctttag atgaactcat tgatagttgt 540 gaaccaacac aagtaaaaca tatgatgcaa gtgatagaac cccagtttca acgagacttc 600 atttcattgc tccctaaaga gttggcactc tatgtgcttt cattcctgga acccaaagac 660 ctgctacaag cagctcagac atgtcgctac tggagaattt tggctgaaga caaccttctc 720 tggagagaga aatgcaaaga agaggggatt gatgaaccat tgcacatcaa gagaagaaaa 780 gtaataaaac caggtttcat acacagtcca tggaaaagtg catacatcag acagcacaga 840 attgatacta actggaggcg aggagaactc aaatctccta aggtgctgaa aggacatgat 900 gatcatgtga tcacatgctt acagttttgt ggtaaccgaa tagttagtgg ttctgatgac 960 aacactttaa aagtttggtc agcagtcaca ggcaaatgtc tgagaacatt agtgggacat 1020 acaggtggag tatggtcatc acaaatgaga gacaacatca tcattagtgg atctacagat 1080 cggacactca aagtgtggaa tgcagagact ggagaatgta tacacacctt atatgggcat 1140 acttccactg tgcgttgtat gcatcttcat gaaaaaagag ttgttagcgg ttctcgagat 1200 gccactctta gggtttggga tattgagaca ggccagtgtt tacatgtttt gatgggtcat 1260 gttgcagcag tccgctgtgt tcaatatgat ggcaggaggg ttgttagtgg agcatatgat 1320 tttatggtaa aggtgtggga tccagagact gaaacctgtc tacacacgtt gcaggggcat 1380 actaatagag tctattcatt acagtttgat ggtatccatg tggtgagtgg atctcttgat 1440 acatcaatcc gtgtttggga tgtggagaca gggaattgca ttcacacgtt aacagggcac 1500 cagtcgttaa caagtggaat ggaactcaaa gacaatattc ttgtctctgg gaatgcagat 1560 tctacagtta aaatctggga tatcaaaaca ggacagtgtt tacaaacatt gcaaggtccc 1620 aacaagcatc agagtgctgt gacctgttta cagttcaaca agaactttgt aattaccagc 1680 tcagatgatg gaactgtaaa actatgggac ttgaaaacgg gtgaatttat tcgaaaccta 1740 gtcacattgg agagtggggg gagtggggga gttgtgtggc ggatcagagc ctcaaacaca 1800 aagctggtgt gtgcagttgg gagtcggaat gggactgaag aaaccaagct gctggtgctg 1860 gactttgatg tggacatgaa ggaattctgc agatatccag cacagtggcg gccgctcgag 1920 tctagagggc ccttcgaagg taagcctatc cctaaccctc tcctcggtct cgattctacg 1980 cgtaccggtc atcatcacca tcaccattga 2010 25 669 PRT Artificial Sequence Description of Artificial Sequence V5HIS tagged homo sapien 25 Met Cys Val Pro Arg Ser Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly Val Leu Leu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys Leu Ser Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40 45 Gly Leu Tyr Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr 50 55 60 Glu Ser Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr 65 70 75 80 Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser 85 90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135 140 Ile Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145 150 155 160 Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu 165 170 175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile 180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile 290 295 300 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp 305 310 315 320 Asn Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr 325 330 335 Leu Val Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn 340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val 370 375 380 Arg Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385 390 395 400 Ala Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val 405 410 415 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg 420 425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln 530 535 540 Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser 545 550 555 560 Ser Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe 565 570 575 Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val 580 585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val 610 615 620 Asp Met Lys Glu Phe Cys Arg Tyr Pro Ala Gln Trp Arg Pro Leu Glu 625 630 635 640 Ser Arg Gly Pro Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly 645 650 655 Leu Asp Ser Thr Arg Thr Gly His His His His His His 660 665 26 2001 DNA Artificial Sequence Description of Artificial Sequence MYCHIS tagged homo sapiens 26 atgtgtgtcc cgagaagcgg tttgatactg agctgcattt gcctttactg tggagttttg 60 ttgccggttc tgctccctaa tcttcctttt ctgacgtgcc tgagcatgtc cacattagaa 120 tctgtgacat acctacctga aaaaggttta tattgtcaga gactgccaag cagccggaca 180 cacgggggca cagaatcact gaaggggaaa aatacagaaa atatgggttt ctacggcaca 240 ttaaaaatga ttttttacaa aatgaaaaga aagttggacc atggttctga ggtccgctct 300 ttttctttgg gaaagaaacc atgcaaagtc tcagaatata caagtaccac tgggcttgta 360 ccatgttcag caacaccaac aacttttggg gacctcagag cagccaatgg ccaagggcaa 420 caacgacgcc gaattacatc tgtccagcca cctacaggcc tccaggaatg gctaaaaatg 480 tttcagagct ggagtggacc agagaaattg cttgctttag atgaactcat tgatagttgt 540 gaaccaacac aagtaaaaca tatgatgcaa gtgatagaac cccagtttca acgagacttc 600 atttcattgc tccctaaaga gttggcactc tatgtgcttt cattcctgga acccaaagac 660 ctgctacaag cagctcagac atgtcgctac tggagaattt tggctgaaga caaccttctc 720 tggagagaga aatgcaaaga agaggggatt gatgaaccat tgcacatcaa gagaagaaaa 780 gtaataaaac caggtttcat acacagtcca tggaaaagtg catacatcag acagcacaga 840 attgatacta actggaggcg aggagaactc aaatctccta aggtgctgaa aggacatgat 900 gatcatgtga tcacatgctt acagttttgt ggtaaccgaa tagttagtgg ttctgatgac 960 aacactttaa aagtttggtc agcagtcaca ggcaaatgtc tgagaacatt agtgggacat 1020 acaggtggag tatggtcatc acaaatgaga gacaacatca tcattagtgg atctacagat 1080 cggacactca aagtgtggaa tgcagagact ggagaatgta tacacacctt atatgggcat 1140 acttccactg tgcgttgtat gcatcttcat gaaaaaagag ttgttagcgg ttctcgagat 1200 gccactctta gggtttggga tattgagaca ggccagtgtt tacatgtttt gatgggtcat 1260 gttgcagcag tccgctgtgt tcaatatgat ggcaggaggg ttgttagtgg agcatatgat 1320 tttatggtaa aggtgtggga tccagagact gaaacctgtc tacacacgtt gcaggggcat 1380 actaatagag tctattcatt acagtttgat ggtatccatg tggtgagtgg atctcttgat 1440 acatcaatcc gtgtttggga tgtggagaca gggaattgca ttcacacgtt aacagggcac 1500 cagtcgttaa caagtggaat ggaactcaaa gacaatattc ttgtctctgg gaatgcagat 1560 tctacagtta aaatctggga tatcaaaaca ggacagtgtt tacaaacatt gcaaggtccc 1620 aacaagcatc agagtgctgt gacctgttta cagttcaaca agaactttgt aattaccagc 1680 tcagatgatg gaactgtaaa actatgggac ttgaaaacgg gtgaatttat tcgaaaccta 1740 gtcacattgg agagtggggg gagtggggga gttgtgtggc ggatcagagc ctcaaacaca 1800 aagctggtgt gtgcagttgg gagtcggaat gggactgaag aaaccaagct gctggtgctg 1860 gactttgatg tggacatgaa ggaattctgc agatatccag cacagtggcg gccgctcgag 1920 tctagagggc ccttcgaaca aaaactcatc tcagaagagg atctgaatat gcataccggt 1980 catcatcacc atcaccattg a 2001 27 666 PRT Artificial Sequence Description of Artificial Sequence MYCHIS tagged homo sapiens 27 Met Cys Val Pro Arg Ser Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly Val Leu Leu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys Leu Ser Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40 45 Gly Leu Tyr Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr 50 55 60 Glu Ser Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr 65 70 75 80 Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser 85 90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135 140 Ile Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145 150 155 160 Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu 165 170 175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile 180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile 290 295 300 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp 305 310 315 320 Asn Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr 325 330 335 Leu Val Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn 340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val 370 375 380 Arg Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385 390 395 400 Ala Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val 405 410 415 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg 420 425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln 530 535 540 Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser 545 550 555 560 Ser Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe 565 570 575 Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val 580 585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val 610 615 620 Asp Met Lys Glu Phe Cys Arg Tyr Pro Ala Gln Trp Arg Pro Leu Glu 625 630 635 640 Ser Arg Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 645 650 655 Met His Thr Gly His His His His His His 660 665 28 25 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 28 tcacttcatgtccacatcaaagtcc 25 29 27 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 29 ggtaattacaaagttcttgttgaactg 27 30 22 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 30 ccctgcaacgtgtgtagacagg 22 31 24 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 31 ccagtctctgcattccacactttg 24 32 23 DNA Artificial Sequence Description of Artificial Sequence Oligonucleotide primer 32 ctcagacaggtcaggacatttgg 23 

What is claimed is:
 1. An isolated human sel-10 polypeptide comprising SEQ ID NO:7.
 2. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:3.
 3. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:4.
 4. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:5.
 5. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:6.
 6. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:8.
 7. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:9.
 8. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:21.
 9. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:25.
 10. The isolated sel-10 polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:27. 